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—^  I 

HICAGO  MEDICAL  SCHOOL  I 
LIBRARY 

Vol. 
<L        n 
Donor 


Date 


.\ 


THE  LIBRARY 

OF 

THE  UNIVERSITY 
OF  CALIFORNIA 

PRESENTED  BY 

PROF.  CHARLES  A.  KOFOID  AND 
MRS.  PRUDENCE  W.  KOFOID 


MEDIUM  CONTINENTAL,  THREE-FIFTHS    ACTUAL  SIZE. 

Jiausch  and  Lomb  Optical  Company. 

A,  base  or  foot;  B,  pillar;  C,  arm,  in  it  the  spring  for  the  fine  adjustment; 
I),  body;  E,  objective;  F,  draw-tube;  G,  eyepiece  or  ocu'ar:  H,  mirror;  I,  sub- 
stage  condenser;  J,  stage;  K,  slide;  L,  fine  adjustu-ent;  M,  coarse  aujuatmenU 


DIRECTIONS 


FOR  WORK  IN 


Histologieal  Laboratory 


FOR  THE  USE  OF 


MEDICAL  CLASSES 


IN  THE 


UNIVERSITY  OF  MICHIGAN. 


G.  CARL  HUBER,  M.  D., 

ASSISTANT    PROFESSOR   OF    HISTOLOGY    AND    EMBRYOLOGY. 


GEORGE  WAHR, 

PUBLISHER  AND  BOOKSELLER, 

ANN  ARBOR,  MICHIGAN. 


COPYRIGHTED 
E 

1892. 


COURIER  PRESS,  ANN  ARBOR. 


B/o  It  w 


v 


"V 

4^ 


^ 


PREFACE. 


The  following  pages  have  been  prepared  with  the  hope  that  they  niay 
be  a  guide  and  a  help  to  the  students  doing  work  in  the  Histological  Labor- 
atory. In  the  very  brief  description  that  is  given  of  the  tissues  to  be  studied 
I  have  born  in  mind  that  the  students  entering  on  this  work,  will  have  had, 
a  course  of  lectures  on  Histology.  The  aim  of  the  notes  is  therefore  not 
to  supplant,  but  rather  to  supplement  the  text-books  on  this  subject. 

Drawings  are  to  be  made  of  nearly  all  the  preparations  to  be  examined. 
There  is  no  better  way  for  the  student  to  obtain  a  correct  and  a  lasting 
impression  of  the  tissues  to  be  studied  than  by  carefully  sketching  what  he 
sees  under  the  microscope.  The  drawings  are  to  be  made  in  the  laboratory. 
A  few  methods  for  hardening,  macerating,  embedding  and  staining  tissues 
are  given ;  such  as  have  proved  themselves  to  be  most  reliable  have  been 
selected.  Anyone  familiar  with  the  methods  here  given  can  without  diffi- 
culty employ  any  he  may  find  recorded  in  the  works  on  microscopical- 
tech  nic. 

I  am  indebted  to  the  Bausch  &  Lomb  Optical  Company  for  sectional 
diagram  of  their  Model  microscope ;  to  Mr.  S.  P.  Budgett  for  the  draw- 
ings of  the  two  wood  cuts.  I  also  wish  to  acknowledge  the  help  received  in 
the  arrangement  of  lessons,  from  Schaffer's  Essentials  of  Histology. 

G.  C.  H. 


£1347970 


SUPPLIES. 


The  student  before  entering  on  the  work  should  supply  himself  with 
the  following  outfit : 

Six  dozen  slides ;  >^  ounce  No.  2  ^-inch  square  cover  glasses  and  2 
dozen  No.  2  |^-inch  circles;  a  slide  box;  a  sharp  razor,  flat  on  one  side;  a 
fine  pair  of  scissors;  2  teasing  needles;  3  solid  watch  crystals;  I  section 
lifter  ;  I  Camels  hair  brush ;  a  hard  and  a  soft  drawing  pencil ;  I  Canada 
halsam  tube ;  and  I  pair  of  cover-glass  forceps. 


BOOKS  OF  REFERENCE. 


Toldt. — Lehrbuch  der  Gewebelehre. 

Behrens,  Kossel,  Schiefferdecker. — Die  Gewebe  des  Menschlichen  Korpers. 
.  Kolliker. — Handbuch  der  Gewebelehre  des  Menschen. 

Schwalbe. — Lehrbuch  der  Anatomic  der  Sinnesorgane. 

Cadiat. — Taite  D  Anatomic  Generale. 

Klein  and  Noble  Smith. — Atlas  of  Histology. 

Qitain. — Elements  of  Anatomy.  Vol.  I.  Part  II.  General  Anatomy 
or  Histology,  by  Prof.  Schaffer. 

Satterthivaite. — Manual  of  Histology. 

Orth. — Normale  Histologie. 

Stohr. — Lehrbuch  der  Histologie. 

Microscopical  Technic. — 

Lee. — The  Microtomist's  Vade-Mecum. 

Whitman. — Methods  of  Microscopical  Anatomy  and  Embryology. 

Behrens. — Tabellen  zum  Gebrauch  bei  Mikroskopischen  Arbeiten. 

Rturvier. — Traite  technique  d'histologie. 

Boehm  and  Oppel. — Taschenbuch  der  Mikroskopischen  Technik. 

Friedlander-Eberth. — Mikroskopische  Technik  zum  Gebrauch  bei  Medi- 
cinischen  und  Pathlogisch-anatomischen  Unterscuchungen. 

Heneage  Gibbes. — Practical  Histology  and  Pathology. 

Kahlden—  Technik  der  Histologischen  Untersuchung  Pathologisch- 
anatomischer  Praparate. 

JRawitz. — ^Leitfaden  der  Histologi&ehen  Untersuchungen. 

Behrens,  Kossel,  Schiefferdecker. — -Vol.  I. 


LESSON   I. 

CELL  AND  CELL  DIVISION, 

(a)  PLANT  CELLS. 

From  one  of  the  layers  of  an  onion  remove  a  small 
st-rip  of  the  film  which  is  found  on  its  inner  surface. 
Spread  out  this  thin  membrane  on  a  slide  in  a  drop  of 
water,  and  cover  with  a  "cover  glass."  In  studying  it 
use  iirst  the  low  power,  and  employ  one  of  the  smaller 
openings  in  the  diaphragm.  Observe  the  large  cells, 
oblong  or  nearly  square  in  shape,  each  bounded  by  a  dis- 
tinct cell  membrane.  Notice  the  round  or  oval  nucleus, 
usually  seen  at  one  end,  or  near  one  of  the  sides  of  the 
cell. 

Carefully  elevate 'the  cover  glass  from  one  edge,  and 
add  a  drop  of  "  LugoPs  solution,"  to  the  water;  observe 
the  staining.  Make  a  sketch  of  a  number  of  the  cells  as 
seen  under  the  high  power. 

(b)  KARYOKINESIS  IN  PLANT  CELLS. 

The  young  and  growing  "tips"  of  an  onion  were 
hardened  in  Fleming's  solution,  embedded  in  celloidin, 
sectioned,  and  the  sections  were  stained  in  Boehmer's 
haematoxylin,  they  were  then  dehydrated  in  alcohol,  and 
are  now  in  oil  of  bergamot.  To  make  a  " permanent 
mount,"  place  a  section  on  a  slide,  remove  excess  of  oil 
by  means  of  a  small  piece  of  filter  paper,  add  a  drop  of 
Canada  balsam,  and  cover  with  a  "  cover  slip."  Sections 
were  made  longitudinally  through  the  "  tip,"  and  under 
low  power  the  parallel  rows  of  cells  will  be  seen.  Even 
with  this  power  it  will  be  noticed  that  some  of  the  nuclei 
stain  much  more  deeply  than  others,  examine  these  with 


the  high  power  and  one  of  the  stages  of  cell  division 
(Karyokinesis)  will  be  recognized.  Observe  the  difference 
in  the  appearance  of  a  u  resting  nucleus  "  and  one  that  is 
in  the  earlier  stages  of  cell  division;  in  the  latter  the 
chromosoma  can  be  clearly  determined,  while  in  the  for- 
mer a  "  chromatic  network  "  is  presented.  Search  for 
cells  in  which  the  chroinosoma  are  arranged  in  the  form 
of  a  monaster,  and  also  such  as  show  the  dlaster  stage. 
You  may  be  able  to  make  out  the  achromatic  spindle. 

Make  a  sketch  of  the  different  stages  of  mitosis  pre- 
sented in  your  preparation,  as  seen  under  high  power. 

'a.)     UNICELLULAR  ORGANISMS. 

In  the  faecal  matter  found  in  the  rectum  of  a  frog,  it 
is  not  uncommon  to  find,  as  parasites,  unicellular  organ- 
isms belonging  to  the  ciliata  or  flagellata.  Place  a  small 
portion  of  the  frecal  matter  on  a  slide  and  add  a  drop  of 
normal  salt  solution  (NaCl  Q.6%  in  distilled  water)  cover 
with  a  slip.  Under  the  microscope  these  little  animals 
will  be  seen  darting  through  the  field  by  means  of  their 
ciliary  movement.  Place  a  small  drop  of  a  one  per  cent, 
osmic  acid  solution  to  one  side  of  the  cover  glass,  (near 
enough  to  touch  the  edge)  and  with  a  piece  of  filter  paper 
held  to  the  opposite  edge  of  the  slip,  carefully  remove, 
by  absorption,  some  of  the  fluid  under  the  cover ;  in  this 
way  some  of  the  osmic  acid  will  be  drawn  under,  and  will 
in  a  few  moments  fix  the  living  cells  suspended  in  the 
fluid.  Note  their  size,  shape  and  the  structure  of  proto 
plasm  and  nucleus. 


DRAWINGS  FOR  LESSON  I. 


DRAWINGS  FOR  LESSON  I 


LESSON    II. 

CELL  AND  CELL  DIVISION  (CONTINUED). 

(a)  TYPICAL  ANIMAL  CELLS. 

A  small  portion  taken  from  the  ovary  of  a  young  frog 
is  to  be  teased  in  normal  salt  solution,  cover  with  a  "  cover 
glass,"  and  examine  with  the  low  power.  Observe  the  large 
spherical  cells,  with  granular  protoplasm  and  a  nucleus 
which  is  only  indistinctly  discernible.  Elevate  the  edge 
of  the  cover  glass,  and  add  a  few  drops  of  a  one  per  cent, 
solution  of  acetic  acid ;  this  "  clears "  the  protoplasm, 
and  now  a  more  careful  study  of  the  nucleus  can  be  made. 

Make  a  sketch  of  several  of  the  ova  as  seen  under  the 
low  power,  after  the  acetic  acid  has  been  added. 

(b)  KAR10KINESIS  IX  ANIMAL  CELLS. 

Fleming  has  long  ago  shown  that  the  salamander 
teslis  is  one  of  the  most  useful  tissues  for  showing  Karyo- 
kinesis  in  animal  cells.  The  testes  were  hardened  in 
"Fleming's  solution,"  embedded  in  paraffin,  sections  were 
cut  and  these  were  fixed  to  a  cover  glass  by  means  of  an 
"  albumen  fixative,"  the  paraffin  was  removed,  they  were 
stained  in  safranin,  dehydrated,  cleared,  and  are  now  in 
xylol.  Bring  a  slide  on  which  has  been  placed  a  small 
drop  of  Canada  balsam,  and  the  preparation  will  be 
mounted  for  you  at  the  "  distributing  table."  In  this  les- 
son only  the  structure  of  the  cells  and  the  different  stages 
of  Karyokinesis  are  to  be  studied.  Sections  need  to  be 
examined  with  a  high  power.  The  great  majority  of  the 
cells  will  be  "resting  cells,"  showing  a  nucleus  possessing 
a  chromatic,  intranuclear  network  and  a  nucleolus^  a  pro- 
toplasm with  an  intracellular  network. 


.     g 

By  moving  the  sections  about,  cells  fixed  in  the  pro- 
cess of  division  will  be  found.  Make  a  sketch  of  a  cell, 
not  in  the  process  of  division,  and  indicate  in  your  draw- 
ing, as  clearly  as  you  can,  the  finer  structure  of  the  proto- 
plasm and  nucleus.  Reproduce  the  different  stages  of 
Karyokinesis  your  specimen  shows. 

(c)    DEMONSTRATION. 

Under  a  one-twelfth  oil  immersion,  will  be  demon- 
strated a  section  taken  from  a  salamander  testis,  fixed  and 
stained  to  show  accessory  nucleus  (Nebenkern)  in  a  rest- 
ing cell.  Also  cells  fixed  in  process  of  division,  showing 
c/iromosoma,  microsoma,  and  the  different  parts  of  the 
achromatic  spindle. 


DRAWINGS  FOR  LESSON  II. 


DRAWINGS  FOR  LESSON  II. 


LESSON    III. 

MAMMALIAN    BLOOD, 

(a)  FRESH  HUMAN  BLOOD. 

Obtain  a  drop  of  blood  by  pricking  a  carefully  cleaned 
finger  with  a  steel  pen,  one  of  the  prongs  of  which  has 
been  broken  off,  quickly  mount  the  drop  on  a  slide,  and 
examine  with  the  high  power.  Observe  that  most  of  the 
red  cells  are  arranged  in  rouleaux,  and  between  these  now 
and  then  a  white  corpuscle  is  seen.  If  your  drop  was 
small,  some  of  the  red  corpuscles  found  in  the  peripheral 
part  of  your  preparation  will  appear  crenated. 

(b)  FRESH  HUMAN  BLOOD  WITH  NORMAL  SALT. 

Obtain  a  very  small  drop  of  blood  as  above  directed, 
mix  it  on  the  slide  with  a  drop  of  normal  salt  solution, 
and  cover.  The  smaller  number  of  corpuscles  will  allow 
of  a  more  earful  study  of  their,  size  and  shape.  Observe 
that  the  red  appear  as  biconcave  circular  discs,  and  are  a 
little  smaller  than  the  majority  of  the  white.  Some  of  the 
red  will  soon  become  crenated. 

Make  a  drawing  of  a  number  of  the  red  cells  as  seen 
on  "the  flat,''  and  a  few  seen  on  the  edge  (profile). 

(c)  FIBRIN. 

Place  a  large  drop  of  blood  on  the  slide,  cover  with  a 
"slip"  and  allow  it  to  clot.  When  clotted,  wash  gently 
with  a  current  of  water,  (by  placing  a  few  drops  on  one 
side  of  the  cover  and  touching  the  opposite  edge  with  a 
piece  of  filter  paper,)  so  that  some  of  the  red  blood  cells 
may  be  removed. 

Examine  now  with  the  high  power,  and  see  whether 
the  delicate,  glistening  strands  of  fibrin  are  noticeable  in 


—  10  — 

the  field.  This  preparation  may  now  be  stained  by  caus- 
ing a  0.5%  solution  of  Methylenblue  to  flow  under  the 
cover  glass.  The  fibrin  filaments  and  the  white  bfood 
cells  will  take  on  the  blue  color.  ' 

(d)  MAMMALIAN  BLOOD  FIXED  WITH  HAYEM'S  SOLUTION. 

Blood  from  the  carotid  artery  of  a  cat  was  allowed  to 
flow  into  a  quantity  of  Hayem's  solution,  the  fixed  elements 
sank  to  the  bottom,  and  in  24  hours  the  supernatant  fluid 
was  decanted,  the  corpuscles  were  then  washed  in  distilled 
water,  and  after  washing  mixed  with  a  small  quantity  of 
gum  glycerin.  A  small  drop  of  the  mixture  is  to  be  taken 
on  a  slide,  and  covered;  this  will  give  you  a  permanent 
mount.  In  it  the  size  and  shape  of  the  red  corpuscles,  as 
well  as  the  structure  of  the  white  blood  cells,  can  be 
studied. 

(e)  DEMONSTRATION. 

In  a  blood  preparation,  fixed  and  stained  according  to 
Ehrlich's  method,  the  "  blood  plates  "  will  be  demonstrated 
under  an  one-twelfth  inch  oil  immersion. 


DRAWINGS  FOR  LESSON  III. 


DRAWINGS  FOR  LESSON  III. 


LESSON    IV. 

AVIAN  AND  AMPHIBIAN  BLOOD, 

(a.)     PIGEON  BLOOD. 

One  of  the  toes  of  a  pigeon  has  been  amputated,  from 
the  flowing  blood  allow  a  drop  to  fall  on  the  slide,  mix 
with  a  drop  of  normal  salt  solution  and  cover  with  a  slip. 
Examine  under  high  power.  Observe  that  the  red  corpus- 
cles are  oval,  somewhat  flattened  and  nucleated ;  note  the 
relative  size  of  red  and  white  cells.  Cause  a  drop  of  a  one 
per  cent,  solution  of  acetic  acid  to  How  under  the  cover, 
and  you  will  notice  that  the  nuclei  in  the  red  and  white 
cells  can  be  more  clearly  seen. 

Make  a  sketch  of  several  fed  and  white  corpuscles,  as 
they  present  themselves  to  you  after  the  acid  has  been 
added. 

(b)  PIGEON  BLOOD  HARDENED  IN   HAYEM'S  SOLUTION. 

A  pigeon  was  bled  into  a  large  quantity  of  Hayem's 
solution,  the  blood  was  hardened  and  washed  as  described 
in  yesterday's  lesson,  and  is  no  in^gum  glycerin.  A 
small  drop  is  to  be  mounted  on  a  slide,  this  will  serve  as  a 
"permanent  mount,"  and  is  to  be  compared  with  the  pre- 
paration of  blood  already  made. 

(c)  AMPHIBIAN  BLOOD. 

A  drop  of  blood  taken  from  the  opened  heart  of  a  frog 
is  placed  on  a  slide  and  mixed  with  a  drop  of  normal  salt 
solution,  cover  with  a  "  slip."  The  red  corpuscles  are 
oval,  somewhat  flattened  and  nucleated  cells,  much  larger 
than  the  white.  Observe  also,  that  when  the  red  are 
viewed  in  profile,  they  show  a  slight  convexity  in  the 
centre.  Add  a  drop  of  acetic  acid  (one  per  cent,  solution), 


—  12  — 

and  the  nuclei  of  the  red  and  white  corpuscles  will  stand 
out  clearly,  in  those  parts  of  the  field  where  the  acid  has 
come  in  contact  with  them. 

Reproduce  some  of  the  red  as  seen  on  "  the  flat,"  and 
others  seen  in  profile,  also  a  number  of  the  white. 

(d)     AMPHIBIAN  BLOOD  FIXED  WITH  HAYEM'S  SOLUTION. 

Blood  cells  of  a  frog  were  hardened  in  Hay  em's  solu- 
tion, and  are  now  in  gum  glycerin,  mount  a  drop  of  the 
mixture.  Compare  the  cat's  and  the  pigeon's  blood  with 
this  preparation  of  frog's  blood  ;  the  two  latter  show 
nucleated  red  cells,  the  former  not.  Note  the  difference 
in  shape  and  size. 


DRAWINGS  FOR  LESSON  IV, 


HW] 


SCHOOL 


DRAWINGS  FOR  LESSON 


LESSON  V. 

WHITE  BLOOD  AND  LYMPH-CORPUSCLES  ANDTHEIR 
AMCEBOID  MOVEMENT. 

(a)     AMOEBOID  MOVEMENT    IN  WHITE  BLOOD  CELLS    OF  A 
FROG. 

A  small  quantity  of  blood  obtained  from  the  heart  of  a 
frog:,  is  mixed  with  a  drop  of  normal  salt  solution  and  cov- 
ered with  a  slip,  to  prevent  evaporation  while  examining, 
the  following  steps  are  taken :  The  cover  glass  is  fixed  by 
means  of  two  or  three  drops  of  melted  paraffin,  so  placed, 
that  after  congealing,  one  half  of  the  drop  will  rest  on  the 
slide,  the  other  on  the  edge  of  the  cover  ;  after  this  step  has 
been  taken,  the  sealing  is  completed  by  means  of  a  camels- 
hair  brush,  which  has  been  dipped  in  vaseline,  and  drawn 
about  the  edge  of  the  cover.  In  examining  this  prepara- 
tion be  very  careful  not  to  bring  the  end  of  the  objective 
in  contact  with  the  vaseline  on  the  slide.  Search  the 
specimen  until  you  find  a  white  blood  cell  of  somewhat 
irregular  shape,  place  it  in  the  centre  of  the  field  and  make 
a  drawing  of  it  as  it  presents  itself  to  you  at  this  time ;  at 
intervals  of  one  or  two  minutes,  carefully  repeat  the 
sketching  until  about  ten  have  "been  made,  you  will  no 
doubt  see  that  the  cell  in  question  has  changed  its  shape 
a  number  of  times,  pseudopodia  may  have  been  thrown 
out  and  again  withdrawn,  etc. 

(1>)    AMOEBOID  MOVEMENT  IN  WHITE  BLOOD  CELLS  OF  CATS. 

The  animal  from  which  this  blood  is  to  be  taken,  was 
severely  bled  24  hours  previously,  a  drop  is  to  be  obtained 
from  a  small  incision  in  the  ear,  the  manner  of  preparing 
and  studying  this  preparation  is  the  same  as  that  describ- 
ed in  the  preceding  paragraph  of  this  lesson. 


— 14  — 

(c)  LYMPH  CORPUSCLES. 

Scrape  the  cut  surface  of  a  fresh  lymphatic  gland  with 
a  scalpel,  and  mix  the  "scrapings"  on  the  slide  with  a 
drop  of  normal  salt  solution.  Cover  and  examine  under 
high  power,  employing  one  of  the  smaller  openings  in  the 
diaphragm,  cells  of  different  sizes  will  be  seen  ;  observe  the 
structure  of  protoplasm  and  nucleus  in  the  different  forms 
presented.  Cause  a  drop  of  a  \%  solution  of  acetic  acid 
to  flow  under  the  cover;  in  a  few  moments  the  nuclei  will 
present  themselves  more  clearly. 

(d)  LYMPH  CORPUSCLES  FIXED  AND  STAINED  WITH  METH  Y- 

LEN  BLUE  AND  EOSIN. 

A  freshly  cut  surface  of  a  lymphatic  gland  was  scrap- 
ed, and  the  scraping  spread,  on  a  cover  glass  ;  fixed  by  ex- 
posing it  to  a  heat  of  110°C,  for  20  minutes,  stained  in 
methylen  blue  and  eosin.  Preparation  will  be  completed 
for  you  at  the  "  distributing  table."  On  examining  with 
the  high  power  the  following  varieties  of  cells  will  be 
seen  in  the  preparation  : 

(1)  Small  lymphocytes — in  size  about  as  large  or  a 
little  larger  than  a  red  blood-cell,  with  a  comparatively 
large  nucleus,  which  takes  the  stain  freely,  and  -only  a 
narrow  border  of  protoplasm.     The  majority  of  the  cells 
will  belong  to  this  order. 

(2)  Large  mononuclear  white  blood  cells — possessing 
a  much  larger  border  of  protoplasm  and  a  nucleus  that 
stains  but  faintly  ;  only  a  few  will  be  found. 

(3)  Ehrlich's     transitional    form — with    horse-shoe 
shaped  nucleus. 

(4)  Poly  nuclear  white  Hood  cells — with  a  lobulated 
nucleus,  staining  very  deeply. 

Make  a  sketch  of  one  cell  from  each  of  the  above 
forms. 

(e)  DEMONSTRATION. 

demonstration  of  white  blood  cells  which  were  stained, 
according  to  Ehrlich^s  methods.  Given  to  show  Eosino- 
phile,  Neutrophile,  and  Basophile  cells. 


DRAWINGS  FOR  LESSON  V. 


DRAWINGS  FOR  LESSON  V 


LESSON  VI. 

EPITHELIUM. 

(a)  PAVEMENT  EPITHELIUM,  ISOLATED  CELLS. 

Scrape  the  inside  of  the  cheek  with  the  edge  of  a  slide 
or  a  clean  knife,  mount  the  scrapings  in  a  drop  of  saliva 
and  cover.  Study  under  high  power  and  make  use  of  one 
of  the  smaller  openings  in  the  diaphragm.  Observe  the 
large,  flat  epithelial  cells  Many  are  single,  others  are 
found  in  groups.  Note  the  round  or  oval  nucleus. 

You  will  also  see  the  '*  salivary  corpuscle,"  (leuco- 
cytes which  have  wandered  through  the  oral  epithelium, 
and  are  swollen  by  the  imbibition  of  water,)  cause  a  drop 
of  methylen  blue  to  flow  under  the  cover,  and  the  staining 
will  bring  to  prominence  the  parts  above  described. 
Reproduce  several  pavement  cells  and  a  salivary  corpus- 
cle, as  seen  under  high  power. 

(b)  CUTANEOUS  EPITHELIUM,  FROM  FROGS  EPIDERMIS. 

It'  a  frog  is  imprisoned  for  some  time  in  a  jar  contain- 
ing a  small  quantity  of  water,it  will  be  noticed  that  portions 
of  the  epidermis  are  from  time  to  time  shed  and  found 
floating  in  the  water.  These  thin  membranes  were  washed, 
stained  in  Boehrner's  hsematoxylin,  dehydrated  in  alco- 
hol, and  are  now  in  oil  of  cloves,  to  complete  the  "  mount," 
place  the  tissue  on  slide,  remove  excess  of  oil,  add  a  drop 
of  Canada  balsam,  and  cover  with  a  "  slip."  This  prepara- 
tion will  give  you  a  surface  view  of  a  stratified  pavement 
epithelium. 

(0     CROSS  SECTION  OF  STRATIFIED  EPITHELIUM. 

A  small  portion  of  the  mucous  membrane  lining  the 
soft  palate  of  a  dog  was  hardened  in  a  saturated  watery 
solution  of  mercuric  bichloride,  stained  in  carmin,  embed- 
ded in  paraffin  and  sectioned. 

The  method  of  mounting  paraffin  sections  is  the  fol- 
lowing :  Come  to  the  distributing  table  with  a  slide  on 


- 16  — 

which  a  thin  layer  of  the  "albumen  fixative"  has  been 
spread,  (place  a  small  drop  of  the  iixative  on  the  centre  of 
a  clean  slide,  spread  it  out  as  thin  as  you  can  with  a  clean 
glass  rod,  then  with  a  dry  and  clean  finger  wipe  away  all 
excess,)  a  section  will  then  be  fixed  to  the  slide  ;  now 
place  slide  on  the  water  bath,  and  allow  it  to  remain  there 
until  the  paraffin  begins  to  melt,  quickly  remove  the 
melted  paraffin,  by  covering  the  section  with  a  few  drops 
of  oil  of  turpentine,  (paraffin  is  soluble  in  oil  of  turpen- 
tine,) remove  excess  of  oil,  and  add  a  drop  of  Canada 
balsam.  Care  should  be  taken  not  to  over-heat  the  prepara- 
tion, while  the  paraffin  is  being  melted,  nor  to  allow  it  to 
dry  after  the  oil  of  turpentine  has  been  removed. 

Observe  the  several  layers  of  epithelial  cells,  and  their 
change  in  shape  as  they  are  traced  outwards.  In  the 
lowest,  the  cells  are  columnar,  in  the  following  polygonal, 
while  in  the  outermost  layer,  they  are  flattened. 

Notice  the  change  in  the  shape  and  structure  of  the 
muclei,  belonging  to  the  cells  of  the  different  layers. 
Make  a  sketch  as  seen  under  high  power. 

(d)  ISOLATED  COLUMNAR  CELLS. 

The  intestinal  canal  of  a  frog  was  dissociated  in  33% 
alcohol  for  18  hours,  fixed  in  osmic  acid,  washed  in  water. 
A  small  portion  of  the  epithelial  lining  is  to  be  teased  in 
gum  glycerin,  and  mounted.  Many  isolated  cells  will  be 
seen  in  the  field,  (use  high  power  and  one  of  the  smaller 
openings  in  the  diaphragm,)  observe  their  shape,  position 
of  oval  nucleus,  and  their  striated  border. 

(e)  GOBLET  CELLS. 

The  mucous  layer  of  the  large  intestine  of  a  cat  was 
macerated  in  33%  alcohol,  and  fixed  in  osmic  acid  (as 
above).  Place  a  small  portion  of  the  tissue  in  a  drop  of 
methyien  blue,  and  allow  to  stain  for  five  minutes,  wash  in 
water,  tease  stained  tissue  in  gum  glycerin,  and  mount. 
You  will  notice  columnar  cells,  also  many  in  which  the 
free,  the  inner  portion  of  the  cell  appears  distended,  (by 
means  of  a  mucigen  which  has  formed  there)  these  are  the 
"goblet  cells."  Sketch  several  columnar,  and  two  goblet 
cells  as  seen  under  the  high  power. 


DRAWINGS  FOR  LESSON  VI, 


DRAWINGS  FOR  LESSON  VI 


LESSON  VII. 

EPITHELIUM  AND  ENDOTHELIUM, 

(a)  ISOLATED  CELLS  OF  CILIATED  EPITHELIUM. 

The  mucous  membrane  of  the  trachea  of  a  calf  was 
placed  in  33%  alcohol  for  12  hours,  then  into  a  \%  sol.  of 
osmic  acid  for  24  hours,  stained  in  methylen  blue  and  is  to 
be  teased  in  gum  glycerin  and  mounted.  Observe  that 
most  of  the  cells  are  columnar  in  shape,  possessing  an  oval 
nucleus.  Notice  particularly  the  cluster  of  fine,  short  hairs 
adhering  to  the  free  end  of  the  cell.  Some  of  the  cells 
may  be  distended  with  mucigen  (goblet  cells),  if  so  no 
ciliary  border  will  be  seen.  Sketch  several  as  seen  under 
the  high  power. 

(b)  CROSS  SECTION  OF  STRATIFIED  COLUMNAR,  CILIATED 

EPITHELIUM. 

The  mucous  membrane  lining  the  palate  of  a  frog,  was 
hardened  in  u  Fleming's  solution/'  stained  in  alum  car- 
min,  embedded  in  paraffin  and  cut.  Steps  for  mounting 
sections  cut  in  paraffin  were  fully  given  in  paragraph  (c) 
of  the  last  lesson.  This  epithelium  is  composed  of  three 
layers  of  cells,  the  cells  of  the  outer  layer  being  ciliated. 

(c)  DEMONSTRATION  OF  CILIARY  MOVEMENT. 

A  small  portion. is  removed  from  the  pharynx  of  a  frog 
and  mounted  in  normal  salt  solution,  the  cover  glass 
should  be  supported  by  means  of  a  thick  hair. 

(d)  TRANSITIONAL  EPITHELIUM. 

The  bladder  of  a  dog  was  injected  with  33%  sol.  of  alco- 
hol, at  the  end  of  four  hours  it  was  cut  into  small  pieces  and 
these  were  allowed  to  remain  in  the  above  "dissociating 
rluid  "  for  12  hours,  were  then  fixed  in  osmic  acid,  stained 


in  inethylen  blue  and  are  to  be  teased  and  mounted  in 
gum  glycerin. 

Observe  the  different  forms  of  the  cells  presented; 
large,  somewhat  flattened  cells,  (depending  on  the  degree 
of  distention)  often  showing  two  nuclei,  these  belong  to 
the  inner  stratum  ;  the  cells  found  in  the  next  two  layers 
are  short  columnar  or  "pear-shaped"  and  often  quite 
irregular.  Make  a  sketch  of  a  number  of  them  as  seen 
under  high  power. 

(c)    ENDOTHELIAL  CELLS. 

A  \%  sol.  of  silver  nitrate  was  injected  into  the  peri- 
toneal cavity  of  a  frog,  after  15  minutes  the  intestines  with 
the  mesentery  were  removed,  the  latter,  while  fixed  in  an 
extended  position,  was  exposed  to  sun  light  until  the  silver 
was  reduced.  Mount  in  gum  glycerin.  Observe  that 
the  silver  is  deposited  in  the  intercellular  cement,  a  deli- 
cate black  line  surrounding  each  cell,  (the  protoplasm  and 
nucleus  often  take  a  light  brown  color). 

Make  a  sketch  of  a  number  of  these  cells  as  seen  under 
high  power. 


DRAWINGS  FOR  LESSON  VII 


DRAWINGS  FOR  LESSON  VII. 


LESEON  VIII. 

CONNECTIVE  TISSUE. 

(a)  FIBRILS  OF  WHITE  FIBROUS  CONNECTIVE  TISSUE. 

A  short  piece  of  a  tendon  was  subjected  to  the  digestive 
action  of  a  glycerin  extract  of  the  pancreas,  at  a  temper- 
ature of  35°  C,  (Evvald  and  Kiihne).  A  small  portion  is  to 
be  teased  in  normal  salt  solution.  If  the  teasing  has  been 
thoroughly  done,  many  isolated  elementary  fibrils  will  be 
seen  in  the  field.  Observe  that  they  do  not  anastomose, 
are  often  somewhat  wavy.  Cause  a  1%  solution  of  acetic 
acid  to  flow  under  the  cover  glass,  and  it  will  be  seen  that 
the  fibrils  swell  up  and  become  homogeneous. 

(b)  FIBRILS  OF  YELLOW  ELASTIC  CONNECTIVE  TISSUE. 

Tease  a  small  portion  of  the  elastic  tissue,  taken  from 
the  ligament  nuchae  of  an  ox,  in  normal  salt  solution. 
Observe  the  fibrils  of  yellow  elastic  tissue,  they  are  highly 
refractive,  branch  and  anastomose,  and  the  broken  ends 
u  curl  back."  Cause  a  few  drops  of  \%  sol.  of  acetic  acid  to 
flow  under  the  cover  glass,  and  notice  that  the  yellow 
elastic  fibrils  are  not  affected  by  it. 

(c )  AREOLAR  CONNECTIVE  TISSUE. 

A  small  portion  taken  from  the  subcutaneous  tissue 
of  a  young  cat  is  placed  on  a  dry  slide,  the  (dues  are  drawn 
out,  and  made  to  adhere  to  the  slide  by  al  owing  them  to 
dry,  while  the  center  is  kept  moist  by  breathing  upon  it. 
Jn  tins  way  a  thin  film  may  be  obtained.  Place  a  drop  of 
normal  salt  on  a  cover  glass  and  invert  it  over  the  center 
of  the  film. 

In  examining  use  the  high  power,  and  employ  one  of 
the  smaller  openings  in  the  diaphragm.  Observe  the  bun- 


dies  of  white  iibrous  connective  tissue,  composed  of  the 
elementary  fibrils,  crossing  the  field  in  different  directions ; 
between  which  fibres  of  yellow  elastic  tissue  will  be  seen, 
having  a  distinct  outline,  giving  and  receiving  branches, 
and  often  possessing  a  straight  course.  Look  for  the  con- 
nective tissue  cells. 

Carefully  elevate  the  cover  glass  and  add  a  drop  of  a 
\%  sol.  acetic  acid.  In  a  few  moments  the  bundles  of 
white  fibrous  tissue  will  swell  and  appear  homogeneous, 
while  the  unaffected  yellow  elastic  fibers  will  stand  out 
boldly;  the  nuclei  of  the  fixed  connective  tissue  cells  can 
now  be  more  clearjy  seen.  Make  a  sketch  of  a  bundle  of 
white  fibrous  tissue,  some  yellow  elastic  fibers,  and  a  num- 
ber of  the  connective  tissue  cells. 

(d)  TENDON. 

Tendons  taken  from  the  tail  of  a  mouse  were  sus- 
pended in  alum  carmin  for  24  hours.  They  are  to  be 
teased  in  gum  glycerin.  The  tendon  fasciculi  are  some- 
what swollen,  between  them  rows  of  tendon  cells,  which 
have  taken  the  red  color  will  be  distinctly  seen,  they  are 
oblong  or  square  in  shape,  with  the  nucleus  usually  at 
one  end. 

Make  sketch  of  two  fasciculi  with  theintervening  cells. 

(e)  CROSS-SECTION  OF  TENDON. 

A  tendon  was  hardened  in  chromic  acid,  cut,  and 
stained  in  acid  fuchsin.  Sections  are  in  oil  of  bergamot, 
and  are  to  be  mounted  in  Canada  balsam.  Examine  with 
the  low  power.  Observe  that  the  tendon  is  surrounded  by  a 
connective  tissue  sheath  composed  of  bundles  of  fibrous  tis- 
sue, the  majority  of  which  run  transversely  ;  from  this  septa 
pass  in,  and  divide  the  tendon  into  larger  and  smaller 
bundles,  these  are  composed  of  tendon  fasciculi,  their  cut 
ends  being  presented  to  you.  Between  the  fasciculi  the 
tendon  corpuscles  will  be  seen. 


DRAWINGS  FOR  LESSON  VIII, 


DRAWINGS  FOR  LESSON  VIII. 


LESSON   IX. 

CONNECTIVE  TISSUE.    (CONTINUED.) 

(a)  CROSS-SECTION  OF  LIGAMENT  NUCHAE. 

A  small  piece  of  the  ligament  nuchae  of  an  ox  was 
allowed  to  dry,  fine  shavings  are  to  be  cut  from  one  end, 
by  means  of  a  sharp  scalpel.  Place  the  section  on  a  slide 
in  a  few  drops  of  water,  stain  for  five  minutes  in  acid 
fnchsin,  wash  in  water  and  mount  in  gum  glycerin. 
Observe  the  cut  end  of  the  elastic  fibres,  arranged  in 
smaller  and  larger  bundles. 

Make  a  sketch  of  a  small  portion  as  seen  under  high 
power. 

(b)  EMBRYONIC  CONNECTIVE  TISSUE. 

Umbilical  cord  of  human  foetus  was  hardened  in 
Miiller's  solution,  stained  in  alum  carmin,  embedded  in 
paraffin,  and  cross-sectioned.  Fix  the  sections  to  the 
slide,  remove  the  paraffin.  On  observing  the  preparation 
the  umbilical  vessels  will  be  seen  in  cross-section,  occu- 
pying the  more  central  part  of  the  section,  in  this  lesson 
however  the  connective  tissue  is  to  receive  special  atten- 
tion. 

Observe  the  branched  connective  tissue  cells,  sending 
out  long  processes.  The  cells  are  separated  by  a  large 
amount  of  a  granular,  intercellular  ground  substance, 
in  which  fine  connective  tissue  fibrils,  as  well  as  the  highly 
refractive  elastic  fibre,  may  be  seen.  Make  a  sketch  as 
seen  under  high  power. 

(c)  FAT  CELLS. 

A  portion  of  the  mesentery  of  a  young  rabbit  is  spread 
out  on  a  slide,  in  a  drop  of  normal  salt  solution.  Examine 

3 


—  22  — 

under  low  power,  and  notice  the  large,  nearly  spherical 
cells,  collected  into  small  groups,  the  lobules,  in  some  of 
the  cells  a  flattened  nucleus  may  be  seen,  it  having  been 
pressed  to  one  side.  Cause  a  drop  of  osmic  acid  to  flow 
under  the  slide  and  notice  how  the  fat  globules  in  the 
cells  are  blackened. 

Make  a  sketch  as  seen  under  low  power. 

(d)     SECTION  OF  ADIPOSE  TISSUE. 

Subcutaneous  tissue,  (from  the  sole  of  foot)  was  hard- 
ened in  10%"  nitric  acid,  washed,  embedded  in  celloidin 
and  sectioned.  Sections  were  stained  inBoehmer's  luematox- 
vlin  and  eosin,  dehydrated  in  alcohol  and  are  now  in  oil  of 
bergamot.  Place  section  on  the  slide,  remove  excess  of 
oil,  and  mount  in  Canada  balsam.  On  studying  this  prep- 
aration you  will  find  groups  of  fat  cells,  separated  by 
bundles  of  connective  tissue,  this  having  been  stained  in 
the  eosin.  All  nuclei  are  blue  having  taken  the  haematox- 
ylin. 

Sketch  a  few  of  the  fat  cells  as  seen  under  the  high 
power. 


DRAWINGS  FOR  LESSON  IX, 


DRAWINGS  FOR  LESSON  IX. 


LESSON  X. 

CARTILAGE, 

(a)  HYALIN  CARTILAGE. 

Remove  by  means  of  a  sharp  razor  a  thin  section  from 
the  articular  surface  of  a  frog's  femur,  and  mount  in  nor- 
mal salt  solution.  Examine  under  high  power,  using  one 
of  the  smaller  openings  in  the  diaphragm. 

Observe  the  round  or  oval  cartilage  cells  each  sur- 
rounded by  a  capsule,  and  possessing  a  large  spherical 
or  oval  nucleus.  The  cells  are  often  found  in  groups  of 
2-4  or  more,  and  are  in  small  spaces  (the  lacunae)  in  the 

homogeneous,  intercellular  ground  substance,  the  matrix. 

\ 

(b)  HYALIN  CARTILAGE.     (STAINED.) 

One  of  the  ring  cartilages,  taken  from  the  trachea  of  a 
young  dog.  was  hardened  in  picric  acid  (saturated  watery 
solution)  embedded  in  celloidin  and  cross-sectioned.  Sec- 
tions were  stained  in  hgemotoxylin  and  eosin,  and  are  now 
in  oil  of  bergamot,  mount  in  Canada  balsam.  Note  the 
perichondrium  about  the  hyalin  cartilage. 

Observe  the  structure  of  the  cartilage  cells,  and  make 
a  sketch  as  seen  under  high  power,  including  in  your 
drawing  a  small  segment  of  the  perichondrium  and  a  por- 
tion of  the  adjoining  hyalin  cartilage. 

(c)  ELASTIC  FIBRO- CARTILAGE. 

The  elastic  cartilage  was  taken  from  a  calf's  ear, 
hardened  in  a  saturated,  watery  solution  of  picric  acid, 
embedded  in  celloidin,  sectioned  and  sections  were  stained 
in  picro-lithion-carmin,  they  are  now  in  oil  of  bergamot, 
mount  in  Canada  balsam. 


-24  — 

Observe  that  in  place  of  the  homogeneous  intercel- 
lular ground  substance,  as  seen  in  hyalin  cartilage,  you 
have  one  that  is  permeated  with  a  well  defined  network  of 
yellow  elastic  fibres. 

Make  a  drawing  as  seen  under  high  power. 

(d)  WHITE  FIBRO-CARTILAGE. 

An  intervertebral  disc  from  a  calf  was  hardened  in  a 
saturated  watery  solution  of  picric  acid,  embedded  in  cel- 
loidin,  sectioned  and  the  sections  were  stained  in  haema- 
toxylin  andeosin;  they  are  now  in  oil  of  bergamot,  mount 
in  balsam. 

In  fibro- cartilage  the  intercellular  ground  substance 
is  composed  largely  of  bundles  of  white  fibrous  tissue, 
between  these  are  found  the  cartilage  cells,  enclosed  in 
their  capsule,  and  often  surrounded  by  a  thin  layer  of  a 
homogeneous,  hyalin  matrix. 

Make  a  sketch  as  seen  under  the  high  power. 

(e)  DEMONSTRATION    OF    THE  LYMPH- C AN ALICULAR  SYS- 

TEM IN  HYALIN  CARTILAGE. 

A  section  taken  from  an  articular  surface,  (from  the 
bone  of  an  ox)  was  placed  in  30%  solution  of  chromic  acid, 
(Henke  and  Budge)  after  2-5  min.,  it  was  washed  in  water, 
then  stained  in  haematoxylin  and  eosin,  and  mounted  in 
gum  glycerin. 


DRAWINGS  FOR  LESSON  X. 


DRAWINGS  FOR  LESSON  X, 


LESSON  XI. 

BONE. 

(a)     CROSS-SECTION  OF  BONE. 

From  a  thoroughly  macerated  and  dried  bone  a  thin 
cross- section  is  removed  from  the  shaft  by  means  of  a  fine 
saw,  this  is  ground  between  two  hones,  until  it  becomes 
very  thin  and  transparent,  care  being  taken  to  keep  the 
stones  well  moistened  with  water,  and  not  to  use  too  much 
pressure.  The  section  is  now  washed,  first  in  distilled 
water  and  then  in  alcohol,  this  is  best  done  by  means  of  a 
fine  camels-hair  brush,  and  in  a  deep  watch  crystal.  In 
order  to  determine  whether  your  preparation  is  clean, 
mount  it  in  a  drop  of  alcohol  and  examine  under  the  micro- 
scope, the  Haversian  canals  and  the  lacunae  must  not  be 
full  of  sand,  as  they  will  be  if  the  section  was  not  well 
washed.  If  clean,  remove  it  from  the  slide,  place  between 
two  pieces  of  filter  paper,  and  allow  it  to  remain  until 
perfectly  dry.  Bring  to  the  table  and  it  will  be  mounted 
for  you  in  <w  hard  balsam." 

Examine  first  under  low  power.  Observe  that  in  a 
"  Haversian  system  "  the  bone  lamellse  are  concentrically 
arranged  about  the  Haversian  canal.  Between  the  lam- 
ellae, note  the  bone  lucunae,  these  communicating  with  one 
another  by  means  of  the  fine  canaliculi.  Look  for  inter- 
stitial and  circumferential  lamellae. 

Make  a  sketch  of  several  Haversian  systems  as  seen 
under  low  power. 

(b)     LONGITUDINAL  SECTION  OF  BONE. 

A  thin  longitudinal  section  is  made  from  the  shaft  of 
a  bone  by  means  of  a  fine  saw,  grind  and  wash  as  cross- 
section,  when  clean  allow  it  to  dry,  and  it  will  be  mounted 
for  you. 


Study  first  under  the  low  power,  and  observe  the 
anastomosing  Haversian  canals,  the  bone  lamellae  and 
lacunae  are  arranged  parallel  to  them. 

Make  the  drawing  from  the  low  power. 

(c)  DECALCIFIED  BONE,  CROSS -SECTION. 

A  small  portion  taken  from  the  shaft  of  a  fresh  bone 
was  hardened  and  decalcified  in  a  solution  of  nitric  and 
hydrochloric  acid,  (IINO^,  10%"  and  HC1  IX,  equal  parts) 
thoroughly  washed  in  flowing  water,  cross- sectioned,  and 
the  sections  were  stained  in  \%  sol.  of  acid  fuchsin.  They 
are  to  be  mounted  in  gum  glycerin. 

Examine  first  under  low  power  and  observe  the  re- 
semblance to  the  section  of  "  hard  bone  ; "  under  high  power 
the  bone  corpuscles  will  be  seen  in  the  lacunae. 

Study  also  the  periosteum. 

(d)  LONGITUDINAL  SECTION  OF  DECALCIFIED  BONE. 

This  was  prepared  as  the  cross-section  and  is  to  be 
mounted  in  gum  glycerin. 

Compare  with  longitudinal  section  of  hard  bone. 


DRAWINGS  FOR  LESSON  XI. 


DRAWINGS  FOR  LESSON  XT. 


LESSON  XII. 

DEVELOPING  BONE  AND  BONE  MARROW, 

(a)  DEVELOPING  BONE. 

One  of  the  developing  bones  removed  from  a  foetal 
limb  was  hardened  and  decalcified  in  picro-suphuric  acid, 
embedded  in  celloidin,  sectioned  and  stained  in  Irnema- 
toxylin  and  eosin.  Sections  are  now  in  oil  of  bergamot, 
and  need  to  be  mounted  in  Canada  balsam. 

Section  is  to  be  studied  first  under  the  low  power,  and 
if  it  comes  from  the  centre  of  the  bone,  it  will  show  the 
following  areas,  named  in  order  as  they  present  them- 
selves when  the  section  is  moved  from  the  articular  surface 
toward  the  middle  of  the  shaft,  where  ossification  is  most 
advanced. 

1).     Area  of  articular  cartilage  ; 

2).     Area  of  flattened  cartilage  cells,  arranged  in  rows; 

3).  Area  of  enlarged  cartilage  cells,  calcification  may 
have  taken  place  in  the  matrix  between  the  rows ; 

4).  Area  of  ossification,  osseous  substance  is  being 
deposited  by  the  "  osteoblasts,"  on  the  calcified  trabecullre 
of  the  cartilage.  In  the  spaces  between  the  trabecullse, 
(the  primary  marrow  spaces)  small  arteriols  and  capil- 
laries, surrounded  by  marrow  cells,  are  found.  The  peri- 
ostial  bone  is  being  formed,  and  is  well  "  marked  off"  from 
the  endochondral  bone. 

Make  a  sketch  as  seen  under  the  low  power,  bringing 
out  in  your  drawing  the  several  areas  mentioned  above. 

(b)  RED  MARROW. 

A  small  portion  of  red  marrow  taken  from  the  femur 
of  a  young  kitten  is  teased  in  normal  salt  solution.  Study 
under  high  power.  Search  the  preparation  for  nucleated 
red  blood  cells. 


—  28  — 

(c)  RED  MARROW— STAINED  AFTER  ARNOLD'S  METHOD. 

Red  manvow  from  a  kitten  was  placed  in  a  test  tube, 
containing  a  small  quantity  of  normal  salt  solution  which 
had  been  colored  with  methylgreen.  In  this  solution  the 
marrow  was  vigorously  shaken  until  the  cells  were  iso- 
lated. Place  a  drop  of  this  fluid,  containing  some  of  the 
isolated  cells,  on  a  slide,  cover  and  examine  under  high 
power.  This  preparation  will  enable  you  to  differentiate 
the  various  kinds  of  cells  normally  found  in  red  marrow. 

Make  a  sketch  of  the  following  types,  as  seen  under 
high  power : 

1).     A  mononuclear  giant  cell  ; 

2).  Polynuclear  giant  cell,  (nucleus  may  be  lobu- 
lated),  S-shaped  or  ring  formed  ; 

3).  Nucleated  red  corpuscle,  with  spherical  nucleus 
which  takes  the  stain  very  freely ; 

4).  Erythroblasts,  a  little  larger  than  nucleated  red 
blood  cells  containing  none  or  very  little  hsemaglobin; 

5).  Various  forms  of  white  blood  cells,  polynuclear, 
transitional,  mononuclear,  and  perhaps  a  few  lymph- 
ocytes; 

6).  Myelocytes,  or  red  marrow  cells,  mononuclear 
cells,  a  little  larger  than  the  white  blood  corpuscles. 

(d)  MARROW  OF  GUINEA  PIG,  STAINED  IN  EHRLICH'S  NEU- 

TROPHILE  MIXTURE. 

Red  marrow  taken  from  the  femur  of  a  Guinea  pig 
was  spread  out  on  a  cover-glass,  fixed  by  exposing  to  a 
heat  of  110°  C.  for  15  min.,  then  stained  in  "  Ehrlich's  neu- 
trophile  mixture."  Bring  to  the  table  a  slide  and  the 
preparation  will  be  mounted  for  you. 

Examine  under  high  power  and  search  for  the  cells 
found  in  bone  marrow. 

A  specimen  will  be  demonstrated  under  the  1-12  inch 
oil  immersion,  notice  the  large  eosinophile  cells,  (with  red 
granulation);  myelocytes,  with  the  neutrophile  granulation. 


DRAWINGS  FOR  LESSON  XII. 


DRAWINGS  FOR  LESSON  XII. 


LESSON  XIII. 

VOLUNTARY  OR  STRIPED  MUSCLE, 

(a)  STRIPED  MUSCLE  OF  FROG,  FRESH. 

Tease  a  small  shred  of  muscle  taken  from  the  leg  of 
a  frog  in  normal  salt  solution.  Study  first  under  low 
power.  Observe  the  long  cylindrical  fibres,  showing  a 
transverse  striation.  Move  the  slide  about  and  you  will 
find  broken  fibres,  the  broken  ends  often  united  by  the 
sarcolemma. 

Cause  a  few  drops  of  acetic  acid  (IX  sol.)  to  flow 
under  the  cover  glass,  and  in  a  few  moments  the  muscle 
nuclei  will  be  seen. 

Make  a  sketch  showing  sarcolemma,  also  a  small 
segment  of  a  muscle  fibre  showing  muscle  nuclei  as  seen 
under  high  power. 

(b)  STRIPED  MUSCLE  OF  CAT. 

A  0.5X  solution  of  osmic  acid  was  injected  into  one 
of  the  skeletal  muscles  of  a  cat,  in  30  minutes  the  muscle 
was  removed,  washed  in  flowing  water,  and  is  to  be  teased 
and  mounted  in  gum  glycerin. 

Observe  the  structure  of  striped  muscle  fibre. 

(c)  BRANCHED  STRIPED  MUSCLE  FIBRES. 

The  posterior,  the  free  end  of  a  frog's  tongue  was 
macerated  for  several  hours  in  u  M.  Schultz'  Mixture,"  a 
small  portion  is  to  be  teased  in  gurn  glycerin. 

When  a  voluntary  muscle  fibre  is  inserted  into  a 
mucous  membrane  or  the  epidermis,  the  end  so  inserted  is 
often  branched. 

This  preparation,  if  you  have  been  careful  in  teasing 
it,  will  show  you  these  branched  fibres. 

Sketch  one  as  seen  under  the  high  power. 

4 


—  30  — 

(d)  CROSS  SECTION  OF  VOLUNTARY  MUSCLE. 

One  of  the  eye  muscles  of  a  dog  was  hardened  in  a 
saturated  watery  solution  of  bichloride  of  mercury,  stained 
in  borax-carmin,  embedded  in  paraffin,  and  cross-sectioned. 
Sections  are  to  be  fixed  to  slide  by  means  of  the  "albumen 
fixative,"  the  paraffin  removed  and  mounted  in  balsam. 

Study  first  under  low  power.  Observing  the  peri  and 
endomysium.  Under  high  power  note  the  structure  of  the 
muscle  fibres,  showing  the  areas  of  Cohnheim.  Observe 
the  position  of  the  muscle  nuclei.  Sketch  as  seen  under 
this  power. 

(e)  INJECTED  VOLUNTARY  MUSCLE. 

Longitudinal  sections  of  an  injected  voluntary  muscle 
which  had  been  hardened  in  alcohol,  and  embedded  in 
paraffin,  were  made.  Fix  sections  to  slide  by  means  of 
the  "  albumen  fixative,"  remove  paraffin  and  mount  in 
Canada  balsam. 

Observe  the  network  of  injected  capillaries.  The 
fibres  are  not  stained.  Sketch  as  seen  under  low  power. 

(f)  DEMONSTRATION  OF  MOTOR  ENDINGS   IN  VOLUNTARY 

MUSCLE. 

Preparation  was  made  from  one  of  the  eye  muscles  of 
a  young  rabbit,  the  method  suggested  by  Ehrlich,  of  in- 
jecting a  1%  solution  of  methylen  blue  into  the  circula- 
tion, was  used.  • 


DRAWINGS  FOR  LESSON  XIII. 


DRAWINGS  FOR  LESSON  XIII 


LESSON  XIV. 

INVOLUNTARY  AND  CARDIAC  MUSCLE, 

(a)  INVOLUNTARY  MUSCLE,  TEASED. 

The  muscular  coat  of  the  small  intestine  of  a  cat  was 
macerated  for  15  minutes  in  a  30%  solution  of  caustic 
potash,  the  tissue  was  then  placed  in  a  saturated  solution 
of  acetate  of  potassium  (to  arrest  the  maceration),  was 
washed  in  water,  and  is  now  in  gum  glycerin.  Observe 
the  long,  fusiform  or  spindle-shaped  cells,  the  protoplasm 
often  showing  a  longitudinal  striation  and  possessing  a 
rod-shaped  nucleus. 

Sketch  several  as  seen  under  high  power. 

(b)  SECTION  OF  INVOLUNTARY  MUSCLE. 

The  muscular  coat  of  the  small  intestine  of  a  cat  was 
hardened  in  absolute  alcohol,  stained  in  borax-carmin, 
embedded  in  paraffin,  and  sectioned.  Fix  the  section  to 
slide  by  means  of  the  albumen  fixative,  remove  the  para- 
ffin and  mount  in  balsam. 

In  your  preparation  the  fibres  in  the  longitudinal  coat 
will  appear  in  cross  section  ;  note  that  they  are  of  different 
sizes,  and  only  a  few  seem  nucleated,  (many  cross- sections 
are  made  of  a  cell,  only  one  of  which  may  pass  through 
the  nucleus). 

The  circular  layer  is  cut  in  the  direction  of  the  long- 
axis  of  the  cells.  Make  a  sketch  as"  seen  under  high  power, 
including  in  your  drawing  a  small  portion  of  both  the 
circular  and  longitudinal  coats. 

(c)  ISOLATED  HEART  MUSCLE  CELLS. 

Small  pieces  of  the  cardiac  muscle  of  a  dog  were  mac- 
erated in  30%  solution  of  KOH  for  15  minutes,  maceration 


—  32  — 

interrupted  by  placing  the  tissue  in  a  saturated  solution  of 
potassium  acetate,  tease  in  gum  glycerin. 

Observe  the  short  oblong  cells,  one  end  being  usually 
branched.  Cells  show  a  cross-striation,  and  possess  one, 
occasionally  two,  oval  nuclei. 

Draw  several  as  seen  under  high  power. 

(d)    SECTION  OF  HEART  MUSCLE. 

The  ventricle  of  a  rabbit's  heart  was  hardened  in  a 
saturated  watery  solution  of  picric  acid,  stained  in  picro- 
carmin,  embedded  in  paraffin  and  sectioned.  Fix  section 
to  slide  and  mount  in  balsam.  Note  how  the  cells  are 
cemented  together  into  fibres,  these  into  bundles. 

Make  drawing  as  seen  under  the  high  power. 


DRAWINGS  FOR  LESSON  XIV. 


DRAWINGS  FOR  LESSON  XIV. 


LESSON  XV. 

MEDULLATED  AND  NONMEDULLATED  NERVE 
FIBRES, 

(a)    MEDULLATED  NERVE  FIBRES. 

Tease  a  piece  from  the  sciatic  nerve  of  a  frog  in  nor- 
mal salt  solution,  before  covering  arrange  the  fibres  as 
straight  as  possible.  Examine  under  high  power,  employ- 
ing one  of  the  smaller  openings  in  the  diaphragm. 

Observe  the  axis  cylinder,  seen  as  a  light  band  pass- 
ing down  through  the  centre  of  the  fibre,  surrounded  by  a 
thin,  glistening  layer,  usually  of  a  light  green  color,  the 
medullary  sheath,  around  this  the  neurolemma.  Find 
the  nodes  of  Ranvier,  search  for  the  nucleus  of  an  inter- 
nodal  segment.  Some  of  the  fibres  may  show  the  seg- 
ments of  Lanterman. 

(I))-    MEDULLATED  NERYE  FIBRE  STAINED  IN  OSMIC  ACID. 

The  sciatic  of  a  frog  was  fixed  in  a  \%  solution  of 
osmic  acid,  tease  very  carefully  in  gum  glycerin.     Exam 
ine  under  high  power. 

The  medullary  sheath  is  stained  deeply  black  by  the 
osmic  acid.  The  u  nodes  of  Ranvier "  are  very  clearly 
seen  in  this  preparation. 

(c)    NONMEDULLATED  FIBRES. 

The  splanchnic  nerves  of  a  dog  were  macerated  in  a 
very  weak  solution  of  chromic  acid  (O.OIX),  tease  and 
mount  in  a  drop  of  methylen  blue.  Examine  under  high 
power.  Among  the  medullated  fibres  a  few  will  be  found 
that  do  not  have  a  medullary  sheath,  showing  no  "  nodes 
of  Ranvier,'7  possessing  numerous  nuclei,  this  often  gives 
them  a  "  beaded  "  appearance,  these  are  the  nonmedul- 
lated  fibres. 


-34- 

Sketch  a  few  nonmeclullated  fibres  as  seen  under  the 
high  power. 

(d)  CROSS  SECTION  OF  A  NERVE  TRUNK. 

A  posterior  tibial  nerve  (human)  was  hardened  in  a 
saturated  picric  acid  solution,  embedded  in  celloidin,  sec- 
tioned and  stained  in  hgematoxylin.  Sections  are  now  in 
oil  of  bergamot,  mount  in  balsam.  Study  first  under  low 
power ;  note  how  the  funiculi  are  held  together  by  a  loose 
connective  tissue,  the  epineurium  ;  in  it  groups  of  fat  cells 
and  the  blood  vessels  of  the  nerve  trunk  are  found.  Each 
funiculus  is  surrounded  by  a  dense  connective  tissue  sheath 
the  perineurium,  this  shows  a  lamellar  structure.  Under 
the  high  power  nerve  fibres  in  cross-section  should  be 
studied.  Between  the  nerve  fibres  of  a  funiculus  a  small 
amount  of  connective  tissue  is  seen,  the  endoneurium. 

Draw  under  low  power  a  number  of  funiculi  and  the 
surrounding  peri  and  epineurium;  under  high  power  a 
small  portion  of  a  funiculus  showing  the  fibres  in  cross- 
section. 

(e)  DEMONSTRATION  OF  THE  FIBRILAE  OF  THE  AXIS  CYLIN- 

DER. 

A  small  nerve  trunk  was  hardened  in  a  IX  solution 
of  osmic  saturated  with  picric  acid,  embedded  in  paraffin, 
sectioned  and  stained  in  Boehmer's  haematoxylin.  The 
preparation  will  be  shown  under  the  one-twelfth  inch  oil 
immersion  and  will  show  the  fibrilae  of  the  axis  cylinder, 
between  which  a  small  amount  of  neuroplasma  is  found. 


DRAWINGS  FOR  LESSON  XV. 


DRAWINGS  FOR  LESSON  XV. 


LESSON  XVI. 

NERVE   CELLS   FROM   SPINAL  CORD,  BRAIN   AND 
PERIPHERAL  GANGLION. 

(a)  NERVE  CELLS  FROM  SPINAL  CORD. 

The  gray  matter  was  dissected  from  the  cervical  cord 
of  an  ox,  macerated  for  several  days  in  a  very  weak  solu- 
tion of  chromic  acid  (1-15,000),  was  then  stained  in 
lithinm-carmin.  Tease  very  carefully  in  gum-glycerin, 
controlling  your  results  under  the  low  power.  Aim  to 
isolate  several  cells  from  the  surrounding  tissue.  The 
worth  of  this  preparation  will  depend  largely  on  the  care 
with  which  you  have  teased  it. 

Examine  under  high  power  and  observe  the  large 
branching  nerve  cells ;  try  to  make  out  the  axis  cylinder 
process.  Make  a  sketch  of  several  cells  as  seen  under  this 
power. 

(b)  NERVE  CELLS  FROM  THE  POSTERIOR  ROOT  GANGLION. 

A  small  piece  of  posterior  root  ganglion  was  fixed  in  a 
a1%  solution  of  osmic  acid,  macerated  in  Ranvier's  alco- 
hol for  several  days,  is  now  to  be  teased  in  a  few  drops  of 
a  \%  solution  of  methylenblue,  in  which  allow  it  to  stain 
five  to  ten  minutes  ;  wash  away  excess  of  stain  with  dis- 
tilled water,  and  mount  in  gum-glycerin,  completing  the 
teasing  in  gum-glycerin  if  necessary. 

Examine  under  high  power,  notice  the  spheriodal  or 
oval  ganglion  cells,  possessing  a  large  spherical  nucleus 
and  nucleolus.  Many  of  the  cells  may  show  the  nucleated 
capsule.  If  you  have  been  careful  in  your  teasing  some 
of  the  cells  will  show  the  axis  cylinder  process,  and  you 
may  see  the  T-shaped  junction  with  the  nerve  fibre. 

Draw  one  cell  as  seen  under  the  high  power. 


-36  — 

(c)  SECTION  OF  POSTERIOR  ROOT  GANGLION. 

The  posterior  root  ganglion  of  a  dog  was  fixed  for 
twenty  four  hours  in  a  10.%"  solution  of  nitric  acid,  then 
hardened  in  Miiller's  solution  for  several  days,  embedded 
in  celloidin  ;  sectioned,  stained  in  a  1%  solution  of  acid 
fuchsin.  Sections  are  now  in  oil  of  bergamot,  mount  in 
balsam. 

Study  first  under  low  power,  noting  the  firm  connec- 
tive tissue  capsule  about  the  ganglion,  continuous  with 
the  epineuriuin  of  the  in-coming  and  out-going  nerve 
trunk.  From  the  capsule  connective  tissue  septa  pass  into 
the  interior  of  the  ganglion. 

Collection  of  nerve  cells  are  seen  between  the  group  of 
nerve  fibres.  Under  high  power  observe  the  structure  of 
the  ganglion  cells,  with  their  nucleated  capsules. 

Make  a  sketch  of  the  ganglion  as  seen  under  the  low 
power. 

(d)  DEMONSTRATION    OF    THE    CORTEX    OF     CEREBRUM, 

CEREBELLUM  AND   SPINAL    CORD,  STAINED  AFTER 
GOLGPS  METHOD. 

Sections  were  made  from  an  embryo  pig,  and  in  them 
only  a  few  of  the  nerve  cells  are  stained. 

Observe  the  pyramidal  cells  of  the  cortex,  Piirkinje's 
cells  in  the  cerebellum,  the  motor  (typus  I  of  Golgi)  and 
the  sensory  (typus  II  of  Golgi)  in  the  spinal  cord. 


DRAWINGS  FOR  LESSON  XVI. 


DRAWINGS  FOR  LESSON  XVI. 


LESSON  XVII. 

SPINAL  CORD  AND  BRAIN. 

(a)     SECTION  OF  SPINAL  CORD. 

A  human  cord  was  hardened  in  Miiller's  solution,  em- 
bedded in  celloidin  and  double  stained  in  nigrosin  and 
eosin.  Sections  are  now  in  tho  oil  of  bergamot,  mount  in 
balsam.  It  will  not  be  possible  for  the  student  to  obtain 
more  than  a  general  idea  of  the  structure  of  the  brain  and 
spinal  cord  in  this  lesson ;  many  sections  which  need  be 
especially  stained  are  required  to  bring  out  the  finer 
anatomy  of  the  central  nervous  system. 

Examine  under  low  power,  and  note  the  arrangement 
of  the  grey  and  white  matter.  The  former  appears  in 
cross-section,  in  form  of  two  crescents,  the  convex  borders 
of  which  are  united  by  a  commissure  composed  of  white 
(anteriorly)  and  gray  (posteriorly)  matter. 

The  anterior  horns  of  the  crescents  are  broader  and 
shorter  and  do  not  come  so  near  to  the  surface  as  the  pos- 
terior. 

The  white  matter  surrounds  the  gray,  and  is  composed 
of  medullated  nerve  fibres,  seen  in  cross-section,  between 
these  fibres  a  small  amount  of  neuroglia  tissue  is  ob- 
served. 

In  the  gray  matter  a  very  fine  network  of  fibres  is 
seen,  composed  of  medullated  fibres,  of  naket  axis  cylin- 
ders, branches  of  nerve  cells,  and  of  neuroglial  tissue.  The 
nerve  cells  are  found  in  groups  in  the  anterior  and  pos- 
terior horn. 

Sketch  a  small  portion  of  the  gray  and  white  matter  as 
seen  under  high  power. 

(]>)    SECTION  OF  CORTEX. 

A  portion  of  the  cortex  taken  from  a  brain  (human) 

5 


-38  — 

was  hardened  in  Miiller's  solution,  embedded  in  celloidin 
sectioned,  stained  in  neutral  carmin  (Fritsch's  method). 
Sections  are  now  in  the  oil  of  bergamot;  mount  in 
balsam.  Under  high  power  the  following  layers  will  be 
"  made  out  "  in  the  cortex  : 

1).  The  outer  or  molecular  layer,  composed  largely 
of  >neuroglial  tissue,  in  it  a  few  small  nerve  cells  and  a 
thin  stratum  of  fine  medullated  fibres,  running  parallel  to 
the  surface,  and  found  just  under  the  pia  mater,  are  seen. 

2  and  3).  Layer  of  small  and  large  pyramidal  cells, 
in  the  former  the  cells  are  small  and  close  together,  in  the 
latter  they  are  larger  and  farther  apart,  there  is,  however, 
no  distinct  boundary  line  between  the  two  layers. 

4).  A  layer  of  small  cells,  some  of  which  are  pyra- 
midal, others  spindle-shaped,  others  multipolar. 

Sketch  a  small  segment  of  the  cortex  as  seen  under 
high  power. 

(c)     SECTION  OF  CEREBELLUM  (HUMAX.) 

Hardened  in  Miiller's  fluid,  embedded  in  celloidin, 
sectioned  and  stained  in  nigrosin  and  eosin.  Sections  are 
now  in  oil  of  bergamot,  mount  in  balsam.  Study  first 
under  low  power,  observing  the  folded  appearance  of  the 
cortex. 

Under  high  power  the  gray  matter  shows  the  following 
layers  in  cross-section. 

1).  An  outer  molecular  layer  composed  largely  of 
neuroglial  tissue,  containing  a  few  small  ganglion  cells. 

2).  Between  the  above  stratum  and  the  third  a  single 
layer  of  large  ganglion  cells,  Purkinje^s  cells  are  found ; 
from  the  base  of  these  cells  an  axis  cylinder  process  is 
given  off;  from  the  opposite  pole  one  or  two  protoplasmic 
processes ;  these  extending  into  the  molecular  layer,  there 
dividing  and  redividing,  until  the  processes  have  the  ap- 
pearance of  a  deer's  antlers.* 

3).  The  granular  layer,  composed  largely  of  round 
and  spindle-shaped  cells,  possessing  comparatively  large 


;  —  39—-      • 

nuclei,  so  that  in  the  section  very  little  but  the  nuclei  will 
be  seen.  The  axis  cylinder  process  of  Piirkinje's  cells 
pass  through  this  layer,  become  medullated  and  are  lost  in 
the  white  substances  found  making  up  the  central  portion 
of  the  fold. 

Sketch   the  cortex  of  the  cerebellum  as  seen  under 
high  power. 


DRAWINGS  FOR  LESSON  XVII, 


DRAWINGS  FOR  LESSON  XVII. 


DRAWINGS  FOR  LESSON  XVII. 


LESSON  XVIII. 

ARTERIES,  VEINS  AND  ADENOID  TISSUE. 

(a)     CROSS  SECTION  OF  ARTERY  AND  VEIN. 

The  posterior  tibial  artery  and  vein  (human)  were 
hardened  in  picric  acid,  embedded  in  celloidin,  sectioned, 
stained  in  Boehmer's  haematoxylin.  Sections  are  now  in 
the  oil  of  bergamot,  mount  in  balsam. 

Observe  first  under  low  power ;  in  the  wall  of  the 
artery  three  coats  are  seen. 

1).  An  inner  coat,  the  "tunica  intima"  consisting 
of  the  following  structures:  A  single  layer  of  endothelial 
cells  lining  the  lumen  of  the  artery;  a  thin  stratum  of 
sub-endothelial  connective  tissue  ;  a  stratum  of  elastic 
tissue,  the  so  called  elastic  intima  or  the  fenestrated  layer 
of  Herile. 

2).  Middle  coat,  the  "  tunica  media"  composed 
largely  of  circularly  disposed  bundles  of  nonstriped 
muscle  tissue,  between  these  thin  films  of  elastic  tissue, 
seen  in  cross- section  as  wavy  lines,  are  found. 

3).  Outer  coat  or  "  tunica  adventitia"  consists  of 
bundles  of  white  fibrous  tissue,  felted  into  a  dense  layer. 
Between  these  bundles  a  few  elastic  fibers  are  seen.  This 
coat  is  continuous  with  the  surrounding  connective  tissue. 

The  wall  of  the  vein  is  not  so  thick,  three  coats  are 
seen,  resembling  in  structure  the  ones  observed  in  the 
artery. 

In  the  intima  of  the  vein  the  elastic  layer  is  not  so 
prominent;  in  the  tunica  media  there  is  relatively  less 
muscular  tissue,  the  bundles  of  which  are  separated  by 
white  fibrous  tissue.  The  adventitia  is  thicker  than  the 
corresponding  coat  in  the  artery.  This  preparation  also 


—  42  — 

shows  in  section  a  number  of  small  arteries  and  veins  in 
the  fibrous  tissue  surrounding  the  larger  vessels. 

Make  a  sketch  of  a  segment  of  the  wall  of  the  artery , 
also  of  a  segment  of  the  wall  of  the  vein,  and  of  a  small 
artery  as  seen  under  low  power. 

(b)  CROSS-SECTION  OF  THE  AORTA. 

The  aorta  of  a  dog  was  hardened  in  picric  acid, 
embedded  in  celloidin,  and  stained  in  lithion-picro-carmin. 
Sections  are  now  in  the  oil  of  bergamot,  mount  in  balsam. 
Study  first  under  low  power.  The  media  is  very  well 
developed,  between  the  muscular  elements,  a  large  amount 
of  elastic  tissue  is  found.  Sketch  as  seen  under  low 
power. 

(c)  CAPILLARIES. 

Study  preparation  of  teased  spinal  cord  for  isolated 
capillaries.  Sketch  several  as  seen  under  high  power. 

(d)  ADENOID  TISSUE. 

The  vermiform  appendix  of  a  dog  was  hardened  in 
alcohol,  stained  in  borax  carmin,  embedded  in  paraffin, 
sectioned.  Fix  sections  to  slide  and  mount  in  balsam. 
Examine  under  high  power.  In  a  section  of  adenoid  tis- 
sue very  little  is  seen  of  the  reticular  network  ;  the  inter- 
stices of  the  retiform  tissue  are  so  full  of  cells  that  it  is 
obscured. 


DRAWINGS  FOR  LESSON  XVIII. 


DRAWINGS  FOR  LESSON  XVIII. 


LESSON    XIX. 

LYMPHATIC  GLAND,  SPLEEN  AND  THYMUS. 

(a)     COMPOUND  LYMPH  GLAND. 

A  lymphatic  gland  was  hardened  in  picric  acid  stained 
in  lithion-picro-carniin,  embedded  in  paraffin,  sectioned. 
Fix  sections  to  slide  and  mount  in  balsam.  Examine  iirst 
under  low  power.  Observe  the  capsule,'  from  which 
trabecullre  pass  into  the  gland,  dividing  its  outer  or  corti- 
cal portion  into  comparatively  large  compartments,  in 
which  the  cortical  nodules  of  adenoid  tissue  are  found. 
On  entering  the  medulla  the  trabecullae  divide  and  anas- 
tomose, the  network  so  formed  has  small  meshes,  in  which 
are  found  the  medullary  cylinders  of  adenoid  tissue.  The 
adenoid  tissue  is  composed  of  a  reticular  frame  work  and 
colls. 

"  The  cortical  follicles  and  the  medullary  cylinders  do 
not  completely  fill  out  the  compartments  made  for  them 
by  the  capsule  and  trabecullse  respectively,  but  a  narrow 
peripheral  zone  of  each  compartment  is  left  free,  this  is  a 
lymph  sinus." — (Klein.)  The  lymph  sinuses  form  an 
anastomosing  system. 

Sketch  a  portion  of  a  gland  as  seen  under  low  power. 

(1>)     RETICULUM  OF  ADENOID  TISSUE. 

A  lymph  gland  was  hardened  in  alcohol,  cut  on  the 
freezing  microtome.  Sections  were  stained  in  Boehmers 
liK'inatoxylin.  They  were  then  transferred  to  a  test-tube 
half  full  of  distilled  water,  in  which  they  were  carefully 
shaken  for  ten  or  fifteen  minutes.  In  this  way  many  of 
the  lymph  cells  are  "  shaken  out "  of  the  reticulum. 
Mount  the  preparation  in  gum  glycerin.  Examine  under 


—  44  — 

high   power,  using  one  of  the   smaller  openings  in  the 
diaphragm. 

A  reticulum  of  very  fine  fibrils  will  be  seen,  fixed 
connective  tissue  cells  are  often  found  on  the  network. 
Sketch  as  seen  under  this  power. 

(c)  SECTION  OF  SPLEEN. 

Small  pieces  from  the  spleen  of  a  dog  were  hardened 
in  bichloride  of  mercury,  stained  in   borax-carmin,  em 
bedded  in   paraffin,  and    sectioned.     Fix   sections   to  the 
slide  ;  remove  paraffin  and  mount  in  balsam. 

Observe  the  capsule  composed  of  connective  tissue 
and  non-striped  muscle  cells.  From  the  capsule,  trabecul- 
lae  pass  into  the  gland,  branching  and  forming  an  anas- 
tomosing network  ;  they  are  in  structure  like  the  capsule. 
Within  the  capsule  two  kinds  of  tissue  are  found,  the 
Malpighian  corpuscle  and  the  spleen  pulp. 

The  former  are  composed  of  adenoid  tissue,  this  is 
usually  found  surrounding  an  artery.  In  the  pulp  a 
frame -work  of  fine  fibrils  and  cells  is  found,  in  the  meshes 
of  which  red  and  white  blood  cells  are  seen. 

Make  a  sketch  of  a  portion  as  seen  under  high  power. 

(d)  DEMONSTRATION  OF  SECTION  FROM  THYMUS  GLAND. 


DRAWINGS  FOR  LESSON  XIX. 


DRAWINGS  FOR  LESSON  XIX. 


LESSON    XX. 

SKIN  AND  ITS  APPENDAGES. 

a)     MACERATED  EPIDERMIS. 

A  small  piece  of  skin  was  macerated  for  several  days 
in  a  0.25  %  solution  of  acetic  acid.  The  epidermis  was  then 
carefully  lifted  from  the  dermis  and  stained  in  Bcehmer's 
haematoxylin.  The  fragments  of  epidermis  are  now  in  oil 
of  bergamot,  mount  in  balsam.  Care  being  taken  to  so 
place  the  tissue  on  the  slide  that  the  under  surface  of  the 
epidermis  is  presented  to  the  observer.  On  studying 
under  low  power  you  will  see  the  depressions  in  the  under 
surface  of  the  epidermis,  into  which  the  papillae  of  the 
true  skin  fit. 

(b)    MACERATED  PREPARATION  OF  THE  DERMIS. 

The  skin  was  macerated  as  above;  on  the  freezing 
microtome,  the  dermis  was  cut  into  quite  thick  transverse 
sections,  these  were  stained  in  Boehmer's  haematoxylin, 
are  now  in  the  oil  of  bergamot;  mount  in  balsam. 

Section  is  given  to  demonstrate  the  papillae  of  the 
true  skin,  in  many  the  capillary  net- work  can  be  made 
out. 

Make  a  drawing  of  a  number  of  papillae  as  seen  under 
the  low  power. 

Cc)     CROSS-SECTION  OF  SKIN,  (HUMAN.) 

A  portion  of  the  skin  removed  from  the  plantar  sur- 
face of  the  foot  was  hardened  for  twenty-four  hours  in 
10%  nitric  acid,  then  in  Muller's  fluid,  embedded  in  celloi- 
din  and  sectioned,  double  stained  in  haematoxylin  and 
eosin.  Sections  are  now  in  the  oil  of  bergamot ;  mount  in 
balsam. 


—  46  — 

Section  is  to  be  studied  first  under  low  power,  and 
the  general  arrangement  of  the  tissues  observed. 

In  the  epidermis  the  following  layers  are  described, 
named  in  order  from  within,  outwards. 

1).  Stratum  Malpighi,  (rete  mucosa)  composed  of 
stratified  pavement  epithelium. 

2).  Stratum  granulosum,  a  narrow  layer,  the  cells  of 
which  contain  keratohyalin  granules  (Waldeyer)  in  their 
protoplasm. 

3).  Stratum  lucidum,  composed  of  horny  cells  the 
outlines  of  which  are  very  indistinct. 

4).     Stratum  corneum,  the  thick  outer  layer. 

Observe  also  the  twisted  portion  of  the  sudoriferous 
glands,  passing  through  the  epidermis. 

In  the  cutis  vera  an  outer  denser  portion,  the  papillary 
layer  is  observed,  composed  of  closely  interwoven  bundles 
of  white  fibrous  tissue,  between  which  a  small  amount  of 
elastic  tissue  is  found. 

This  layer  is  beset  with  papillae. 

In  the  deeper  portion  of  the  corium,  the  reticular 
layer,  the  bundles  of  fibrous  tissue  are  loosely  woven. 

In  the  large  open  meshes  groups  of  fat  cells  and  the 
secreting  portion  of  the  sweat  glands  are  found. 

Some  of  the  preparations  may  also  show  a  Yater- 
Pacinian  corpuscle  in  section. 

Make  a  sketch  of  the  several  layers  of  the  epidermis 
and  dermis  as  seen  under  low  power. 

(d)    CROSS-SECTION  OF  THE  SCALP. 

A  portion  of  the  scalp  was  hardened  in  Miiller's  iluid, 
embedded  in  celloidin,  sectioned  and  stained  in  hsema- 
toxylin  and  eosin.  Sections  are  in  oil  of  bergamot ; 
mount  in  balsam. 

In  this  preparation  especial  attention  is  to  be  given 
to  the  study  of  the  hair  and  its  follicle.  An  attempt  was 
made  to  cut  them  longitudinally. 

Examine  first  under  low  power,  and  observe  the  sev- 


—  47  — 

eral  layers  of  the  follicle,  the  sebaceous  gland  in  connec- 
tion with  it,  and  the  arrectores  pili. 
Sketch  as  seen  under  this  power. 

(o)    TANGENTAL  SECTION  OF  SCALP. 

Tissue  was  hardened  in  Mailer's  fluid,  sections  were 
double-stained  in  haematoxylin  and  eosin  ;  are  now  in  oil 
of  bergamot;  mount  in  balsam. 

In  this  preparation  the  hair  follicles  are  seen  in  cross- 
section.     Study  under   high  power.     Each  follicle  shows 
the  following  layers,  named  in  order  from  without,  in : 
In  the  dermal  coat: 

1).     A  longitudinal  fibrous  layer. 
2).     A  circular  fibrous  layer. 
3).     Glassy  layer. 
In  the  epidermal  coat : 

1).    Outer  root  sheath  (rete  mucosa). 
2).    Henle's  layer. 
3).    Huxley's  layer. 
4).    Cuticle  of  the  shaft. 
Sketch  one  follicle  as  seen  under  high  power. 

(f)  DEMONSTRATION  OF  MEISSNER'S  TACTILE  CORPUSCLE, 

In  a  preparation  stained  with  chloride  of  gold. 

(g)  DEMONSTRATION  OF  LONGITUDINAL  SECTION  THROUGH 

THE  THIRD  PHALANX  OF  A  TOE, 

Showing  nail  in  position. 


DRAWINGS  FOR  LESSON  XX, 


DRAWINGS  FOR  LESSON  XX. 


DRAWINGS  FOR  LESSON  XX. 


LESSON  XXI. 

TONGUE,    TASTE-BUDS,    SALIVARY    GLANDS. 

(a)  CROSS-SECTION  OF  TONGUE. 

A  cat's  tongue  was  hardened  in  picric  acid,  embedded 
in  celloidin,  sectioned  and  stained  in  lithion-picro-carmin. 
Sections  are  in  the  oil  of  bergamot,  mount  in  balsam. 
Preparation  is  given  to  show  the  structure  of  the  papillae 
on  the  dorsum  of  the  tongue,  and  the  arrangement  of  the 
muscle  fibres  in  the  tongue.  The  lingual  artery  and  nerve 
will  be  seen  in  cross-section.  Sketch  a  number  of  the 
papillae  as  seen  under  low  power. 

(b)  STRUCTURE  OF  TASTE-BUDS. 

The  papillae  foliata  of  a  rabbit  was  hardened  in  Flem- 
ing's solution,  stained  in  alum-carmin  and  embedded  in 
paraffin.  Fix  sections  to  slide,  and  mount  in  balsam.  The 
sections  were  cut  at  right  angles  to  the  transverse  ridges 
found  in  the  papilla  foliata.  In  the  epithelium  lining  the 
furrows  between  the  ridges,  the  taste  buds  are  embedded. 
In  the  muscular  and  connective  tissue  below  the  papilla 
foliata,  small  serous  glands  will  be  seen  in  section. 

Sketch  two  ridges,  with  the  intervening  furrow,  (in  the 
walls  of  which  the  taste-buds  are  found,)  as  seen  under 
high  power. 

(c )  SUBKAXILLARY  GLAND. 

Submaxillary  gland  of  a  dog  was  hardened  in  absolute 
alcohol,  stained  in  Haidenhain's  haematoxylin,  embedded 
in  paraffin,  and  sectioned.  Fix  sections  to  slide  and  mount 
in  balsam. 

Under  low  power  observe  how  the  acini  are  grouped 
into  lobules,  these  are  loosely  bound  together  by  connec- 


tive  tissue.  Inter  and  intra-lobular  ducts  will  be  seen  in 
section  showing  the  striated  epithelium.  Under  high  power 
the  structure  of  the  alveoli  is  to  be  carefully  studied.  They 
are  surrounded  by  a  membrana  propria,  and  nearly  filled 
with  clear  mucous  cells,  the  nucleus  of  which  is  found  in 
their  peripheral  portion.  Many  of  the  acini  will  show  a 
crescent  of  more  deeply  stained  cells,  the  demilunes  of 
Haidenhain  or  crescents  of  Guianuzzi,  between  the  mucous 
cells  and  the  membrana  propria. 

Sketch  an  intra-lobular  duct  with  a  number  of  the  sur- 
rounding alveoli  as  seen  under  high  power. 

^d)    PAROTID  GLAND. 

Pieces  from  parotid  gland  of  a  dog  were  hardened  in 
absolute  alcohol,  stained  in  Haidenhain's  hoematoxylin, 
embedded  in  paraffin.  Fix  the  section  to  slide  and  mount 
in  balsam. 

Under  low  power  the  same  general  structure  as  ob- 
served in  studying  sub-maxillary  gland  will  be  seen.  Under 
high  power  the  acini  are  found  to  be  a  little  smaller,  and 
are  lined  by  a  cubical  or  polyhedral  cells,  showing  a  gran- 
ular protoplasm. 

Sketch  a  few  alveoli  as  seen  under  high  power. 

(e)    OESOPHAGUS. 

The  oesophagus  of  a  dog  was  hardened  in  a  10  X  nitric 
acid  for  twenty-four  hours,  then  in  Miiller's  fluid  for  several 
days.  Embedded  in  celloidin,  cross-sectioned  and  stained  in 
Boehmer's  haematoxylin  and  eosin,  are  now  in  oil  of  berga- 
mot.  Mount  in  balsam.  Study  first  under  low  power  and 
the  following  coats  will  be*  observed.  The  oeesophagus  is 
lined  by  a  stratified  pavement  epithelium,  this  resting  on 
a  papillated  mucosa,  which  is  limited  externally  by  a  mus- 
cularis  mucosa.  Then  follows  the  sub-mucosa,  a  fibrous 
tissue  coat,  containing  the  larger  vessels.  Your  section 
may  show  mucous  glands  in  this  stratum. 

Next  a  muscular  coat,  composed  of  inner  circular  and 


-51- 

outer  longitudinal  bundles.  If  section  is  from  the  upper 
third  the  muscular  tissue  is  largely  of  the  striped  variety, 
below  this  non-striped. 

Sketch  a  segment  of  the  wall  as  seen  under  low  power. 


DRAWINGS  FOR  LESSON  XXI« 


DRAWINGS  FOR  LESSON  XXI. 


DRAWINGS  FOR  LESSON  XXI. 


LESSON  XXII. 

INTESTINAL  CANAL, 

[a)     CROSS-SECTION   OF  THE    WALL    OF    CARDIAC    END    OF 
STOMACH. 

A  segment  from  the  wall  of  the  cardiac  end  of  the 
stomach  of  a  dog  was  hardened  in  absolute  alcohol,  stained 
in  borax  carmin,  embedded  in  paraffin,  and  sectioned.  Fix 
sections  to  slide  and  mount  in  balsam. 

The  study  of  the  intestinal  canal  will  b£  much  simpli- 
fied, ii  the  student  bears  in  mind,  that  through  its  whole  ex- 
tent( excepting  the  oesophagus,  and  the  lower  part  of  the  rec- 
tum, where  no  outer  serous  coat  is  found)  it  is  composed  of 
four  coats.  The  outer  three  of  which,  namely  :  the  serous  or 
peritoneal,  the  muscular,  (composed  of  an  outer  longitudi- 
nal and  an  inner  circular  layer),  and  the  sub-mucosa,  or  the 
connective  tissue  coat,  are  very  similar  in  structure.  The 
structural  differences  observed  in  the  several  anatomical 
divisions  of  it,  are  found  in  the  mucosa,  and  especially  in 
the  glands  of  this  coat. 

Study  the  sections  first  under  low  power,  the  finer  de- 
tails being  made  out  under  the  high.  The  simple  or  com- 
pound tubular  glands  found  in  the  mucosa  of  the  cardiac 
end  of  the  stomach,  show  a  short  duct  and  a  comparatively 
long  secreting  tubule.  In  the  latter  two  kinds  of  cells  are 
found,  the  central  or  chief  cells,  short  columnar  or  polyhe- 
dral in  shape,  lining  the  lumen,  and  the  parietal  or  oxyntic 
cells  of  Langley,  of  oval  shape. 

Make  a  sketch,  reproducing  the  several  coats,  as  seen 
under  lower  power. 
(I))     MUCOUS  MEMBRANE  OF  CARDIAC  END. 

The  mucous  membrane  of  the  cardiac  end  of  the 
stomach  was  hardened  in  absolute  alcohol,  stained  in 


—  54- 

Haidenhain's  haematoxylin,  embedded  in  paraffin  and 
cross-sectioned.  Fix  to  slide  and  mount  in  balsam.  Study 
under  high  power.  In  the  preparation  the  structure  of  the 
cardiac  glands  can  be  more  easily  made  out,  the  chief  cells 
having  taken  a  steel  gray  color,  while  the  oval  cells  are 
stained  black. 

Sketch  one  gland  as  seen  under  high  power. 

(c)  PYLORIC  END  OF  STOMACH. 

The  tissue  was  taken  from  the  pyloric  end  of  a  dog's 
stomach,  hardened  in  alcohol,  stained  in  borax  carmin, 
embedded  in  paraffin.  Fix  sections  to  slide  and  mount  in 
balsam.  Observe  that  in  the  pyloric  glands  the  duct  is 
longer,  the  secreting  tubule  correspondingly  shorter,  and 
lined  by  only  one  kind  of  cells,  these  resembling  the  chief 
cells  in  the  cardiac  glands.  Your  section  may  show  small 
masses  of  adenoid  tissue,  found  in  the  deeper  portion  of 
the  mucosa. 

Sketch  two  pyloric  glands  as  seen  under  low  power. 

(d)  SMALL  INTESTINE. 

The  small  intestine  of  a  dog  was  hardened  in  absolute 
alcohol,  stained  in  borax  carmin,  embedded  in  paraffin, 
mount  in  balsam. 

Two  specimens  are  given,  one  of  which  contains  a  sol- 
itary gland  in  section.  Note  the  villi,  conical  projections 
of  the  mucosa,  covered  by  a  single  layer  of  columnar  cells. 
Observe  the  chyle  vessel  in  the  center  of  each  villus.  In 
the  mucosa  are  found  many  tubular  glands,  the  crypts  of 
Lieberkiihn,  coming  to  the  surface  between  the  villi.  In 
the  section  showing  the  solitary  gland  the  lymphoid  tissue 
is  found  in  submucosa  and  mucosa. 

Sketch  a  small  segment  of  the  section  not  containing 
the  solitary  gland  as  seen  undergo w  power,  reproducing  in 
your  drawing  the  several  coats. 

(e)  INJECTED  SMALL  INTESTINE. 

The  mesenteric  artery  of  a  cat  was  injected  with  gela- 


ii   OO      ™ 

tin-carmin,  hardened  in  alcohol,  embedded  in  paraffin  and 
sectioned,  mount  in  balsam.  The  section  is  not  stained. 
Observe  under  low  power,  and  note  the  arrangement  of  the 
injected  vessels  in  the  muscular  coat,  in  the  submucosa, 
about  the  crypts  of  Lieberktihn,  and  in  the  villi. 

(f)    DEMONSTRATIONS    OF   AUERBACH'S    AND    MEISSNER'S 
PLEXUS  IN  THE  RECTUM  OF  A  FROG. 

Methylenblue,  (Ehrlich.) 


DRAWINGS  FOR  LESSON  XXII. 


DRAWINGS  FOR  LESSON  XXII. 


DRAWINGS  FOR  LESSON  XXII. 


LESSON  XXIII. 

LARGE  INTESTINE,  LIVER  AND  PANCREAS, 

(a)  LARGE  INTESTINE. 

Large  intestine  of  dog  was  hardened  in  alcohol,  stained 
in  borax-carmin,  embedded  in  paraffin,  and  cross-sectioned 
mount  in  balsam.  Notice  that  in  the  large  intestine  no 
villi  are  found.  Simple  tubular  glands,  the  crypts  of 
Lieberkiihn,  (lined  by  a  single  layer  of  short  columnar 
cells,  the  great  majority  appearing  as  goblet  cells,)  placed 
vertically  in  the  mucosa  are  observed. 

Sketch  the  section  as  seen  under  high  power. 

(b)  LIVER  CELLS. 

Small  pieces  from  the  liver  of  a  cat,  were  macerated  for 
twenty -four  hours  in  Ranviers  alcohol,  for  two  hours  in 
0.5X  osmic  acid.  Tease  in  gum-glycerin.  Study  under 
high  power.  The  liver  cells  are  polyhedral  in  form,  pos- 
sessing a  distinct  intra-cellular  network.  Fat  globules, 
(stained  black,)  may  be  seen  in  the  cells.  As  a  rule  a  sin- 
gle spherical  nucleus  is  found. 

Sketch  several  as  seen  under  high  power. 

(c)  INJECTED  LIVER. 

The  liver  of  a  pig  was  injected  through  the  portal  vein 
with  Barlin-blue,  hardened  in  alcohol,  stained  in  borax- 
carmin,  embedded  in  paraffin  and  sectioned;  mount  in 
balsam.  Study  under  high  power.  Note  that  the  gland 
is  composed  of  lobules,  the  injected  inter-lobular  branches 
of  the  portal  vein,  are  seen  between  them.  From  these 
inter-lobular  vessels  capillaries  pass  into  the  lobule,  uniting 
in  an  intra-lobular  vessel,  these  empty  into  sublobular 
veins.  The  liver  cells  appear  arranged  in  columns  be- 
tween the  capillaries. 
7 


—  SB- 
Sketch  two  lobules  showing  the  blood  supply  as  seen 
under  low  power. 

(d)  SECTION  OF  LIYER. 

Pieces  from  the  liver  of  a  cat  were  hardened  in 
Muller's  fluid,  embedded  in  celloidin,  stained  in  Boehmer's 
hagmatoxylin.  Sections  are  in  oil  of  bergamot,  mount  in 
balsam.  Study  first  under  the  low  power  and  observe  the 
arrangement  of  the  liver  cells  in  the  hepatic  lobules. 
Search  for  a  bile  duct  and  note  that  it  is  lined  by  columnar 
epithelium. 

(e)  LIVER  WITH    BILE  CAPILLARIES  STAINED.     (OPPEL'S 

METHOD.) 

Small  pieces  of  liver  were  hardened  in  a  solution  of 
bichromate  of  potassium  and  osmic  acid  (Ramon  J.  Cajal,) 
for  three  days,  were  then  transferred  to  0.  75%  solution 
of  nitrate  of  silver,  in  which  they  remained  for  several 
days.  Sections  are  in  turpentine,  mount  in  hard  balsam. 
The  bile  capillaries  are  stained  black.  Two  sections  are 
g'ven,  one  from  the  liver  of  an  embryo  rat,  in  this  the  com- 
pound tubular  character  of  the  gland  is  easily  made  out: 
the  second  from  the  liver  of  a  young  kitten,  showing  an 
apparent  network  ot  bile  capillaries  between  the  liver 
cells. 

(f)  '  PANCREAS. 

The  pancreas  of  a  dog  was  hardened  in  bichloride  of 
mercury,  stained  in  borax-carmin,  embedded  in  paraffin. 
Fix  sections  to  slide  and  mount  in  balsam.  Study  under 
high  power.  The  structure  of  the  pancreas  is  in  many  re- 
spects similar  to  that  of  a  serous  gland.  Note  the  inner 
granular  zone  in  the  cells  lining  the  alveoli,  the  nucleus 
is  found  in  the  outer  zone. 

Sketch  several  alveoli  as  seen  under  high  power. 


DRAWINGS  FOR  LESSON  XXIII. 


DRAWINGS  FOR  LESSON  XXIII, 


LESSON  XXIV. 

ORGANS  OF  RESPIRATION, 

(a)  TRACHEA. 

The  trachea  of  a  dog  was  hardened  for  twenty-four 
hours  in  a  10%  nitric  acid  solution,  then  in  Miiller's  fluid 
for  eight  days ;  embedded  in  celloidin  and  cut  longitudi- 
nally. Sections  were  stained  in  hgematoxylin  and  eosin, 
are  now  in  oil  of  bergamot,  mount  in  balsam.  The  trachea 
is  lined  by  stratified,  columnar,  ciliated  epithelium,  resting 
on  a  basement  membrane.  The  rnucosa  is  composed  of 
loose  areolar  tissue,  limited  externally  by  an  elastic  layer. 
In  the  fibrous  layer  several  of  the  cartilagenous  hoops  are 
seen  in  cross-section.  Sections  of  small  mucous  glands 
may  be  observed  in  this  layer. 

Sketch  as  seen  under  high  power. 

(b)  LUNG. 

The  lung  of  a  cat  was  hardened  in  picric  acid,  embed- 
ded in  celloidin,  stained  in  Boehmer's  haematoxylin  and 
eosin.  Sections  are  now  in  the  oil  of  bergamot,  mount  in 
balsam.  Study  first  under  low  power  and  notice  the 
bronchi,  the  blood  vessels  and  the  alveoli.  The  sections  of 
the  large  bronchi,  show  a  lining  of  stratified,  columnar,  cili- 
ated epithelium,  resting  on  a  fibrous  tissue  mucosa  in 
which  small  mucous  glands  may  be  observed  ;  next  a  band 
of  non-striped  muscle  tissue,  outside  of  which  cartilage- 
nous  plates  may  be  seen  in  cross  section.  The  smaller 
bronchi  are  lined  by  a  single  layer  of  cilated  columnar 
cells,  and  the  cartilagenous  plates  are  wanting.  Try  and 
make  out  the  capillaries  between  the  alveoli,  a  few  blood 
cells  may  be  seen  in  them. 


Make  a  sketch  of  a  segment  from  the  wall  of  a  large 
bronchus,  and  of  a  small  bronchial  with  a  few  of  the  sur- 
rounding alveoli,  as  seen  under  high  power. 

a)     INJECTED  LUNG. 

The  lung  of  a  cat  was  injected  through  a  pulmonary 
artery,  hardened  in  alcohol,  embedded  in  paraffin.  Fix  the 
sections  to  the  slide  and  mount  in  balsam.  The  sections 
are  not  stained.  Under  high  power  observe  the  injected 
capillary  network  about  the  alveoli. 

(d)    THYROID  GLAND. 

The  thyroid  gland  was  hardened  in  absolute  alcohol, 
embedded  in  celloidin,  and  stained  in  lithion-picro-carmin. 
Sections  are  in  oil  of  bergamot,  mount  in  balsam. 

The  gland  is  surrounded  by  a  fibrous  tissue  capsule. 
Oval  or  round  alveoli,  lined  by  a  single  layer  of  cubical 
cells,  are  seen  in  section. 

Sketch  a  number  of  alveoli  as  seen  under  high  power. 


DRAWINGS  FOR  LESSON  XXIV. 


DRAWINGS  FOR  LESSON  XXIV 


LESSON  XXV. 

KIDNEY,  ADRENAL  GLAND,  BLADDER, 

(a)  ISOLATED  TUBULES  OF  KIDNEY. 

The  kidney  of  a  small  mammal  was  macerated  for 
twenty-four  hours  in  30%  solution  of  hydrochloric  acid, 
washed  for  an  hour  in  flowing  water. 

Tease  very  carefully  in  gum-glycerin. 

Different  portions  of  the  uriniferous  tubules  will  be 
seen  in  the  field. 

Sketch  a  Malpighian  corpuscle,  a  convoluted  tubule,  a 
loop  of  Henle  and  a  collecting  tubule  as  seen  under  low 
power. 

(b)  LONGITUDINAL  AND  TRANSVERSE  SECTION  OF  KIDNEY. 

The  kidney  of  a  young  rat  (or  other  small  mammal), 
was  hardened  in  alcohol,  stained  in  borax-carmin,  embedded 
in  paraffin.  Fix  sections  to  slide  and  mount  in  balsam. 
Two  sections  are  given,  the  one  was  cut  longitudinally  the 
other  transversely.  Study  under  low  power  and  observe  the 
arrangement  of  the  tubules  in  (he  cortical  and  medullary 
portion,  as  seen  both  in  the  longitudinal  and  transverse 
section.  In  the  preparation  before  you  a  single  Malpi- 
ghian pyramid  is  found. 

In  the  medullary  portion  the  tubules  have  a  more  or 
less  straight  direction,  radiating  from  the  apex  toward  the 
base  of  the  Malpighian  pyramid.  In  the  cortex,  bundles 
of  straight  collecting  tubules  arranged  in  the  form  of  pyra- 
mids, (the  pyramids  of  Ferrein  or  medullary  rays)  the 
bases  of  which  rest  on  the  base  of  the  Malpighian  pyramid, 
are  observed.  Between  the  medullary  rays,  the  labyrinth 
of  the  kidney,  composed  of  the  Malpighian  corpuscles, 


proximal  and  distal  convoluted,  spiral  and  zigzag  portions 
of  the  uriniferous  tubules,  is  found. 

Under  high  power  the  form  and  structure  of  the  epi- 
thelium lining  the  different  segments  of  the  uriniferous 
tubules  is  to  be  studied. 

Sketch  a  portion  of  the  cortex  as  seen  under  low 
power. 

(c)  HORIZONTAL  SECTION  OF  CORTEX. 

The  kidney  of  a  dog  was  hardened  in  Mulleins  fluid, 
stained  in  alum-carmin,  embedded  in  paraffin  and  cut  at 
right  angles  to  the  medullary  rays.  Fix  to  slide  and 
mount  in  balsam.  Under  low  power  the  medullary  rays 
will  be  recognized,  as  round  or.  oval  masses,  composed  of 
quite  small  regular  tubules  seen  in  cross-section.  These 
are  surrounded  by  the  labyrinth. 

Sketch  one  medullary  ray  and  a  number  of  the  con- 
voluted tubules  about  it  as  seen  under  low  power. 

(d)  INJECTED  KIDNEY. 

The  kidney  of  a  dog  was  injected  with  Berlin  blue 
through  the  renal  artery,  hardened  in  alcohol,  stained  in 
borax  carmin,  embedded  in  paraffin.  Fix  sections  to  slide 
and  mount  in  balsam.  Study  under  low  power,  observing 
the  injected  inteiiobular  arteries.  The  glomeruli  with 
their  afferent  and  efferent  vessels,  the  capillary  network 
about  the  convoluted  tubules,  and  the  straight  capillaries, 
of  the  medulla. 

(e)  ADRENAL  BODY. 

The  adrenal  body  of  a  rabbit  was  hardened  in  a  satu- 
rated solution  of  bichloride  of  mercury,  stained  in  borax- 
carmin,  embedded  in  paraffin  and  cross-sectioned.  Fix 
the  sections  to  slide,  and  mount  in  balsam.  Study  first 
under  low  power,  observing  the  arrangement  of  the  cells 
in  the  cortex  and  medulla. ,  Under  high  power  the  struc- 
ture of  the  cells  is  to  be  made  out. 

Sketch  a  portion  of  the  medulla  and  cortex  as  seen 
under  low  power. 


(f)    BLADDER. 

The  bladder  of  a  dog  was  partially  distended  and 
hardened  for  twenty-four  hours  in  a  10,%"  nitric  acid  solu- 
tion, then  in  Miiller's  fluid  for  several  days,  embedded  in 
celloidin,  cross-sectioned,  and  stained  in  hgematoxylin  and 
eosin.  Sections  are  in  oil  of  bergamot;  mount  in  balsam. 
The  epithelium  lining  the  bladder  is  transitional,  and  is  in 
structure  like  that  lining  the  ureter  and  pelvis  of  the 
kidney.  The  epithelium  rests  on  a  fibrous  tissue  mucosa. 
The  muscular  tissue  of  the  bladder  is  non  striped. 

Make  a  sketch  of  a  portion  of  the  preparation  show- 
ing the  several  coats. 


DRAWINGS  FOR  LESSON  XXV, 


DRAWINGS  FOR  LESSON  XXV, 


DRAWINGS  FOR  LESSON  XXV. 


LESSON  XXVI. 

MALE  AND  FEMALE  GENERATIVE  ORGANS, 

(a)  CROSS-SECTION  OF  TESTIS. 

The  testis  of  a  rat  was  hardened  in  a  saturated  watery 
solution  of  picric  acid,  embedded  in  celloidin,  cross-sec- 
tioned and  stained  in  lithium  carmin.  Sections  are  now 
in  oil  of  bergamot;  mount  in  balsam.  The  epididymis  is 
included  in  this  preparation.  Study  first  under  low  power, 
and  observe  the  fibrous  tissue  capsule,  the  tunica  albuginea 
suiTO'tmdi-ng  the  gland,  from  this  fine  septa  pass  into  the 
parenchyma,  supporting  the  seminiferous  tubules  ;  some 
of  these  are  cut  transversely,  others  obliquely,  and  some' 
longitudinally.  The  seminiferous  tubules  are  lined  by 
several  layers  of  epithelial  cells,  the  tubules  of  the  epi- 
didymis by  a  layer  of  columnar  ciliated  cells. 

This  preparation  is  given  to  show  the  general  struc- 
ture of  the  gland. 

Sketch  a  portion  of  the  testis  as  seen  under  low  power. 

(b)  SECTION  OF  TESTIS  TO  SHOW  SPERM  ATOGENESIS. 

Small  pieces  from  the  testis  of  a  guinea-pig  were 
hardened  in  Fleming's  solution,  embedded  in  paraffin.  The 
sections  were  fixed  to  a  coverglass,  double  stained  in  saf- 
rinin  and  licht  grim.  The  coverglasses  to  which  the 
preparation  are  fixed  are  now  in  xylol ;  mount  in  balsam. 
Study  under  high  power,  and  on  moving  the  section  about 
you  will  observe  that  the  variously  cut  seminiferous 
tubules,  show  different  stages  of  development.  In  a  rest- 
ing tubule,  within  the  tunica  propria  are  seen  several 
layers  of  cells;  in  the  outermost  of  which  are  found  the 
spermatogones  (parent  cells),  and  the  lower  portion  of 


the  supporting  cells  of  Sertoli.  Then  comes  a  layer  of 
quite  large  cells  the  spermatocytes  (mother  cells),  descen- 
dants of  the  spermatogones.  Lining  the  lumen  of  the 
tubules  are  found  several  layers  of  small  cells,  the  sperma- 
toblasts  (daughter  cells).  The  mother  cells  dividing  by 
indirect  cell  division  give  rise  to  the  small  inner  cells, 
from  them  the  spermatozoa  are  developed.  Search  for  a 
tubule  showing  the  fusion  of  the  spermatoblasts  with 
the  supporting  cells  of  Sertoli.  Other  tubules  will  show 
the  successive  steps  in  the  development  of  the  spermatozoa 
in  these  cells,  until  they  are  found  free  in  the  lumen  of 
the  tubule. 

Bring  out  in  several  drawings,  made  of  tubules  show- 
ing different  stages  of  development,  the  several  steps  of 
spermatogenesis  as  seen  in  your  preparation. 

(c)    CROSS-SECTION  OF  OYAEY. 

The  ovary  of  a  bitch  was  hardened  in  a  10X  HNO8 
solution  for  twenty-four  hours,  in  Mullet's  fluid  for  several 
days,  embedded  in  celloidin,  stained  in  haematoxylin  and 
eosin.  Sections  are  now  in  oil  of  bergamot;  mount  in 
balsam.  Study  first  under  low  power.  The  ovary  is  cov- 
ered by  a  single  layer  of  germinal  epithelium.  A  medul- 
lary portion  (in  which  large  vessels  are  found)  surrounded 
by  a  cortical  portion  containing  the  Graafian  folliciles 
seen  in  different  stages  of  development,  is  observed.  The 
framework  or  stroma  of  the  ovary  is  fibrous  and  non- 
striped  muscle  tissue.  A  fully  developed  Graafian  follicile 
shows  the  following  structure:  The  surrounding  stroma 
is  denser;  the  follicile  is  lined  by  the  membrana  granu- 
losa,  composed  of  several  layers  of  small  cells.  In  one 
portion  of  the  folliciles  the  membrana  granulosa  is  thick- 
ened, the  discus  proliferous^  in  this  discus  the  ovum  is 
embedded.  The  cavity  of  the  follicle  is  filled  by  the 
liquor  folliculi.  Other  follicles  in  different  stages  of 
development  will  also  be  observed ;  in  some  the  ovum  is 
surrounded  by  only  a  single  layer  of  cells ;  in  others  by 


—  67  — 

several  layers ;  while  in  still  others  the  cavity  of  the  folli- 
cle may  just  be  forming. 

Make  a  sketch  of  a  number  of  follicles  showing  dif- 
ferent stages  of  development  as  seen  under  the  high 
power. 

(d)    UTERUS. 

The  uterus  of  a  pig  was  hardened  in  alcohol,  stained 
in  carmin,  embedded  in  paraffin,  and  cross-sections  of  one 
of  the  horns  were  made.  Fix  sections  to  slide,  and  mount 
in  balsam.  Study  under  high  power.  The  cavity  of  the 
uterus  is  lined  by  a  single  layer  of  ciliated  columnar  epi- 
thelial cells.  The  mucosa  is  quite  thick,  shows  many  small 
vessels  in  section  ;  in  it  the  utricular  glands  are  found;  the 
muscular  tissue  is  non  striped;  observe  the  arrangement 
of  the  bundles. 

Sketch  a  portion  of  the  wall  showing  the  several  coats 
as  seen  under  low  power. 


DRAWINGS  FOR  LESSON  XXVI. 


DRAWINGS  FOR  LESSON  XXVI. 


DRAWINGS  FOR  LESSON  XXVI. 


LESSON  XXVII. 

THE  EYE. 

^)    SECTION  THROUGH  ANTERIOR  HALF  OF  EYE. 

The  eye  (human)  was  hardened  in  10%  solution  of 
HNO3  for  48  hours,  in  Miiiler's  fluid  for  one  week,  the  ante- 
rior half  was  embedded  in  celloidn,  sectioned  and  stained 
in  haematoxylin  and  eosin.  Sections  are.  in  oil  of  ber- 
gamot  mount  in  balsam.  This  section  shows  the  anterior 
portion  of  the  three  coats  of  the  eye  in  their  normal  rela- 
tive position,  and  also  the  lens  and  its  suspensory  liga- 
ment. Study  first  under  low  power. 

The  cornea  is  composed  of  five  layers,  named  in  order 
from  before  backwards:  (1)  Stratified  pavement  epithe- 
lium ;  (2)  BowmanTs  layer  or  the  anterior  homogeneous 
lamella ;  (3)  Substantia  propria,  the  thickest  of  the  sev- 
eral coats,  composed  of  bundles  of  white,  fibrous  tissue 
arranged  in  layers,  between  which  the  corneal  corpuscles 
are  found ;  (4)  The  posterior  elastic  or  Descement's  mem- 
brane ,  (5)  The  eridothelium  of  the  anterior  chamber. 

In  the  sclera  the  bundles  of  white  fibrous  tissue  are 
densely  woven,  and  are  continuous  with  the  fibrous  tissue 
bundles  of  the  substantia  propria  of  the  cornea,  but  are  not 
so  regularly  arranged.  Observe  the  canal  of  Schlemm  as 
seen  in  cross-section  in  the  scelro  corneal  junction.  Of  the 
middle  layer  the  iris,  ciliary  body  and  anterior  portion  of  the 
choroid  are  included  in  this  section.  The  iris  is  covered  an- 
teriorly by  a  layer  of  cells  continuous  with  the  ones  found 
on  the  posterior  surface  of  the  cornea.  The  Stroma  is  a 
loose  fibrous  tissue  in  which  pigmented  and  unpigmented 
branched  cells  and  many  vessels  are  found.  The  iris  is 


—  70  — 

covered  posteriorly  by  a  double  layer  of  deeply  pigmented 
cells,  the  parj  retince  iridis.  The  fibres  of  the  sphincter  of 
the  iris  are  seen  in  cross-section  near  its  free  edge  and  pos- 
terior surface.  Observe  the  lig  amentum  pectinatum  (com- 
posed of  trabecullae  of  fibrous  tissue)  uniting  the  ciliary 
body  to  the  outer  coat  at  the  sclero  corneal  junction.  The 
spaces  between  the  trabecullre  of  this  ligament  communi- 
cate with  the  anterior  chamber  and  are  known  as  Fonta- 
na's  spaces.  The  ciliary  ~body  is  a  very  much  thickened 
portion  of  the  middle  coat,  is  continuous,  anteriority  with 
the  iris  and  posteriority  with  the  choroid.  It  is  composed 
of  meridionally  placed  folds,  the  ciliary  processes  and  the 
ciliary  muscle:  The  meridional  fibres  of  this  muscle  have 
their  origin  from  the  wall  of  the  canal  of  Schlemm  and 
the  adjacent  fibrous  tissue,  extending  backward  to  the 
anterior  portion  of  the  choroid,  (the  tensor  choioidecTe,) 
and  into  the  ciliary  body.  The  equatorial  or  the  circular 
fibres  of  Miiller,  are  observed  in  cross-section  near  the 
ba'se  of  the  iris.  The  ciliary  body  and  processes  are  cov- 
ered by  a  double  layer  of  pigmented  cells,  the  pars  ciliaris 
retinae.  Only  a  small  portion  of  the  choroid  is  seen  in 
this  section.  Note  how  vascular  it  is.  In  the  stroma 
many  branched  and  pigmented  connective  tissue  cells 
are  seen. 

Notice  how  the  coats  of  the  retina  are  quite  abruptly 
reduced  to  a  double  layer  of  cells  at  the  ora  serata,  continu- 
ing over  the  ciliary  body  and  posterior  surface  of  the  iris  as 
pars  ciliaris  retinas  and  pars  iridis  retinae.  The  lens  is 
enclosed  in  a  homogeneous  capsule.  The  substantia  pro- 
pria  of  the  lens  consists  of  Jong,  nucleated  cells  (the  lens 
fibres)  arranged  in  layers.  The  section  may  show  the 
single  layer  of  short  cubical  cells,  found  on  the  anterior 
surface  of  the  lens  just  under  the  capsule.  Observe  the 
suspensory  ligament  of  the  lens,  composed  of  homogene- 
ous fibres,  these  seemingly  arising  from  the  apexes  of  the 
ciliary  processes  and  passing  from  these  to  the  equator  of 
the  lens,  some  uniting  with  the  capsule  of  the  lens  on  its 


—  71  — 

anterior,  others  on  its   posterior  surface.     Make  a  draw- 
ing of  this  preparation  as  seen  under  the  low  power. 

(b)  LENS  FIBRES. 

A  lens  was  macerated  for  several  days  in  0.5,%  solu- 
tion of  I1C1.  Tease  and  mount  in  gum  glycerin,  and 
examine  under  the  high  power. 

Sketch  several  fibres  as  seen  under  this  power. 

(c)  RETINA. 

A  hardened  retina  was  stained  in  haematoxylin,  em- 
bedded in  paraffin,  and  cross-sectioned.  Fix  sections  to 
slide  and  mount  in  balsam.  Study  under  the  high  power 
The  following  layers  are  made  out,  named  in  order  from 
before  backwards.  (1)  Internal  limiting  membrane;  (2) 
layer  of  nerve  fibres;  (3)  layer  of  ganglion  cells;  (4) 
inner  granular  or  molecular  layer;  (5)  the  inner  nuclear 
layer;  (6)  Outer  granular  or  molecular  layer ;  (7)  Outer 
nuclear  layer;  (8)  Outer  limiting  membrane;  (9)  layer 
of  rods  and  cones;  (10)  layer  of  pigment  cells,  this  layer 
often  remains  attached  to  the  choroid  arid  may  therefore 
not  show  in  your  section). 

Sketch  the  retina  as  seen  under  high  power. 

(d)  DEMONSTRATION. 

A  section  through  the  fovea  centralis,  from  human 
eye. 

(e)  DEMONSTRATION. 

Section  through  the  coats  of  an  eye  at  the  point  of 
entrance  of  the  optic  nerve. 


DRAWINGS  FOR  LESSON  XXVII, 


DRAWINGS  FOR  LESSON  XXVII. 


DRAWINGS  FOR  LESSON  XXVII. 


LESSON   XXVIII. 

COCHLEA  AND  OLFACTORY  MUCOUS  MEMBRANE. 

(a)    COCHLEA  OF  GUINEA  PIG. 

The  cochlea  of  a  Guinea  pig  was  hardened  in  Flem- 
ing's solution,  decalcified  in  1%  chromic  acid  embed- 
ded in  celloidin.  Sections  were  cut  in  a  direction  par- 
allel to  the  long  axis ;  were  stained  in  hsematoxylin  and 
acid  fichsin  and  are  now  in  oil  of  bergamot,  mount  in 
balsam.  The  cochlear  canals  were  opened  on  one  side  so 
that  the  hardening  fluid  might  penetrate  more  easily. 
Study  first  under  low  power.  Observe  the  bony  axis,  the 
the  modeolus,  about  which  the  cochlear  canal  is  spirally 
wound.  Sections  of  it  are  seen  on  each  side  of  the  modeolus. 
The  cochlear  canal  is  divided  into  two  portions  by  the  lam- 
ina spiralis  (a  bony  crest  attached  to  the  modeolar  wall  of 
the  canal)  and  the  lasilar  membrane,  extending  from  the 
lamina  spiralis  to  the  ligamentum  spiralis.  The  upper 
portion  of  the  canal  is  the  scala  vestibuli,  the  lower  the 
scala  tympani;  they  are  lined  by  endothelial  cells.  A  tri- 
angular canal,  the  cochlear  duct  or  scala  media  is  cut 
off  from  the  scala  vestibuli  by  Reissner's  membrane.  In 
the  cochlear  duct  resting  on  the  basilar  membrane  is  found 
the  organ  of  Corti,  in  which  the  following  parts  are  to  be 
distinguished  :  The  pillars  of  Corti,  arranged  in  the  form 
of  an  arch ;  the  inner  and  the  three  or  four  outer  hair 
cells ;  Deiter's  cells  supporting  the  outer  hair  cells  ;  periph- 
eral to  the  organ  of  Corti ;  Hensen's  cells,  in  these  cells 
fat  granules  are  found  in  the  guinea  pig.  Note  the  mem- 
brana  tectorium  resting  on  the  organ  of  Corti.  Observe 
the  spiral  ganglion  in  the  lamina  spiralis,  from  this 
nerve  fibres  pass  toward  the  organ  of  Corti  and  make  con- 


—  74  — 

nection  with  the  hair  cells.     They  maybe  seen  passing 
through  the  tunnel  of  Corti. 

Sketch  the  preparation  as  seen  under  low  power,  and 
a  cochlear  duct  with  the  organ  of  Corti  as  seen  under  the 
high. 

(b)  OLFACTORY  MEMBRANE  (TEASED). 

The  olfactory  membrane  of  a  frog  was  fixed  and 
macerated  in  a  0.5%  solution  of  osmic  acid.  Place  a  small 
piece  of  the  macerated  tissue  on  a  slide  and  cover  with  a 
few  drops  of  methylenblue,  allow  it  to  stain  for  several 
minutes,  wash  away  excess  of  stain  with  distilled  water, 
add  a  drop  of  gum-glycerin,  tease  and  mount.  Study  under 
high  power  and  search  for  olfactory  and  sustentacular 
cells. 

Sketch  a  number  of  the  olfactory  cells  as  seen  under 
this  power. 

(c)  OLFACTORY  MEMBRANE. 

The  mucous  membrane  was  removed  from  the  septum 
of  a  rabbit's  nose,  hardened  in  Fleming's  solution,  stained 
in  haematoxylin  and  embedded  in  paraffin;  fix  sections  to 
the  slide  and  mount  in  balsam. 

This  preparation  shows  olfactory  and  respiratory 
mucous  membrane.  The  epithelium  covering  the  latter  is 
stratified,  ciliated,  columnar,  in  the  regio  olfactoria  the  two 
kinds  of  cells  studied  in  the  teased  preparation  will  be 
observed.  In  the  mucosa,  which  is  composed  of  loose 
fibrous  tissue,  Bowman's  glands  will  be  seen  in  section. 

Sketch  as  seen  under  the  high  power. 


DRAWINGS  FOR  LESSON  XXVIIL 


DRAWINGS  FOR  LESSON  XXVIII. 


LESSON   XXIX.* 

TEETH, 

(a)  TOOTH. 

By  means  of  a  fine  saw  a  longitudinal  section  is  cut 
from  a  tooth  (incisors  or  canines  are  best).  Grind  this  on 
an  emerj^-wheel  as  thin  as  you  can,  then  between  two 
hones  until  it  becomes  quite  transparent.  Care  should  be 
taken  to  grind  it  evenly,  while  doing  so  keep  the  hones 
well  moistened  with  water.  Wash  thoroughly  first  in 
water,  then  in  alcohol ;  then  place  the  section  between 
filter-paper  until  perfectly  dry.  Sections  will  be  mounted 
for  you  in  hard  balsam.  The  preparation  is  first  to  be 
studied  under  a  simple  lens,  observing  the  shape  and  size 
of  the  pulp  cavity,  the  relative  proportion  of  the  dentine, 
cement  and  enamel.  The  structure  of  these  parts  is  then 
to  be  studied  under  the  low  power.  In  the  enamel  note 
the  enqmel  prisms.  In  the  dentine  the  dentinal  tubules 
radiating  from  the  pulp  cavity,  and  in  its  peripheral  por- 
tion the  interglobular  spaces.  The  cement  shows  the 
structure  of  bone,  very  seldom,  however,  showing  Haver- 
sian  canals. 

Sketch  the  tooth  as  seen  under  the  simple  magni- 
fier. 

(b)  SECTION  OF  TOOTH,  IN  SITU. 

The  anterior  portion  of  the,  lower  jaw  of  a  dog  was 
hardened  in  HNO3 ,  a  10%  solution,  for  24  hours,  in 
Muller's  fluid  for  several  weeks  ;  was  then  decalcified  in  a 
solution  composed  of  equal  parts  of  a  10%  HNO3  and  a  \% 
HOI.  The  decalcified  tissue  was  embedded  in  celloidin, 
sectioned,  and  sections  stained  in  hsematoxylin  and  eosin, 


*The  following  lessons  are  especially  arranged  for  the  students  in  the 
Dental  Department. 


—  76  — 

and  are  now  in  oil  of  bergamot,  mount  in  balsam.  The 
preparation  shows  the  tooth  in  longitudinal,  the  jaw  in 
cross-seel  ion.  and  is  given  to  show  the  attachment  of  the 
root  of  the  tooth  to  the  wall  of  the  alveolus,  by  means  of 
the  dental  periosteum.  The  different  parts  of  the  tooth 
will  be  recognized.  The  pulp  is  composed  of  a  loose  con- 
nective tissue,  in  it  a  number  of  small  vessels  may  be 
observed  in  section;  on  the  surface  of  the  pulp  a  layer  of 
odontoblasts  is  seen. 

Sketch  the  preparation  as  seen  under  low  power. 

(c)    CROSS -SECTION  OF  DECALCIFIED  TOOTH. 

A  human  tooth  was  hardened  and  decalcified  in  Pere- 
neyi's  fluid,  embedded 'in  celloidin,  cross-sectioned,  double 
stained  in  hasmatoxylin  and  acid  fuchsin.  Sections  are 
now  in  oil  of  bergamot,  mount  in  balsam. 

The  preparation  is  given  to  demonstrate  the  structure 
of  the  pulp.  Study  under  high  power.  Observe  the  loose 
connective  tissue,  consisting  of  a  few  fibrils  and  branched 
connective  tissue  cells.  A  layer  of  odontoblasts  bounds 
the  pulp,  these  cells  have  a  body  somewhat  columnar  in 
shape,  from  this  two  kinds  of  branches  are  given  off; 
pulpal  processes  communicating  with  the  branched  cells 
of  ihe  pulp  and  tubular  processes,  which  enter  the  den- 
tinal  tubules. 

Sketch  as  seen  under  the  high  power,  a  portion  of  the 
pulp  with  odontoblasts  and  a  segment  of  the  adjacent  den 
tine. 


DRAWINGS  FOR  LESSON  XXIX, 


DRAWINGS  FOR  LESSON  XXIX. 


LESSON  XXX. 

DEVELOPING  TEETH. 

A  number  of  preparations,  showing:  several  stages  in 
the  development  of  a  mammalian  tooth  will  be  given.  The 
difficulty  to  obtain  material  showing  DEFINITE  stages,  pre- 
vents a  full  description  of  the  sections  to  be  studied  in  this 
lesson.  The  student  will  need  to  rely  on  the  notes  taken 
at  the  time. 

NOTES. 


DRAWINGS  FOR  LESSON  XXX. 


DRAWINGS  FOR  LESSON  XXX, 


DRAWINGS  FOR  LESSON  XXX. 


METHODS 

FOR 

LABORATORY  WORK 

IN 

HISTOLOGY. 


METHODS  FOR  MACERATING. 


RANYIER'S  ALCOHOL. 

Place  small  pieces  of  the  tissue  to  be  macerated  in 
33%  alcohol,  in  which  they  remain  from  12  to  24  hours, 
then  transfer  them  to  a"0.25X^smic  acid  solution  for  two 
to  four  hours.  They  can  now  be  teased. 

This  method  is  very  useful  for  macerating  epithelial 
tissue,  for  instance,  the  cells  lining  the  intestinal  canal  or 
the  trachea,  for  isolating  the  cells  of  the  liver,  etc. 

CAUSTIC  POTASH. 

Make  a  30%  solution  of  KOH  in  water,  small  pieces 
of  the  tissue  are  placed  in  this  solution  for  about  15  min- 
utes. The  maceration  is  then  interrupted  by  transferring 
the  tissue  to  a  saturated,  watery  solution  of  acetate  of 
potash,  (it  takes  about  60  parts  of  the  acetate  of  potash  to 
saturate  40  parts  of  water),  to  which  a  few  drops  of  glacial 
acetic  acid  have  been  added,  (the  author  adds  five  to  six 
drops  to  25  cc.  of  the  saturated  solution).  In  about  30 
minutes  the  tissue  is  ready  for  teasing;  it  can,  however, 
be  kept  a  long  time,  several  months,  in  the  acetate  of 
potash. 

This. method  is  used  in  macerating  non-striped  and 
heart  muscle,  also  epithelial  cells. 

HYDROCHLORIC  ACID. 

A  30%  watery  solution  is  used,  in  it  the  tissues  remain 
for  12  to  15  hours,  are  then  washed  in  flowing  water  for 
half  an  hour,  they  may  then  be  teased.  HC1  is  especially 
useful  for  isolating  the  tubules  of  the  kidney.  The  macer- 
ated pieces  of  kidney  may  be  placed  in  a  test  tube  contain- 


ing  a  2  %  watery  solution  of  bismark  brown,  in  this  they 
are  shaken  for  10  to  15  minutes  ;  they  are  at  the  same  time 
stained  and  isolated.  Allow  the  test  tube  to  stand  for  a 
few  moments,  the  isolated  tubules  can  then  be  pipetted 
from  the  bottom. 

NITRIC  ACID.     [Gage.] 

A  30%  watery  solution  is  used,  small  pieces  of  the 
tissue  remain  in  the  fluid  about  24  hours,  and  are  then 
thoroughly  washed  in  water. 

Striped  muscle  fibres  are  well  macerated  by  this 
method. 

SULPHURIC  ACID.     [M.  Schultz  and  Ranvier.J 

Place  the  tissue  to  be  macerated  on  the  slide,  add  a 
few  drops  of  the  strong  sulphuric  acid,  and  cover  with  the 
cover-glass.  In  a  few  moments  the  cells  can  be  separated 
by  gently  tapping  or  pressing  with  a  needle  the  top  of  the 
cover-glass. 

Useful  for  macerating  hair,  nail  and  horny  epidermis. 

SCHULTZE'S  MIXTURE. 

One  or  two  grams  of  crystals  of  chlorate  of  potash  are 
mixed  with  a  little  HNO3 ,  only  enough  acid  is  used  to 
make  a  thick  paste.  In  this  the  tissue  to  be  macerated  is 
embedded  for  two  or  three  hours.  At  the  end  of  which 
time  the  tissue  may  be  removed  to  a  slide  and  teased.  If 
it  does  not  tease  easily,  embed  again  in  the  paste,  repeat- 
ing at  intervals  of  about  thirty  minutes,  until  it  teases 
readily. 

This  method  brings  out  nicely  the  branched  muscle 
fibres.  A  frog's  tongue  may  be  used. 

OSMIC  ACID. 

A  l.OX  watery  solution  of  osmic  acid  is  very  useful  in 
macerating  retina,  olfactory  membrane,  etc.  The  tissues 
remain  in  the  fluid  from  24  to  48  hours,  and  may  then  be 
teased. 


-S3  — 

ACETIC  AND  CHROMIC  ACID.     [Arnold.] 

Tissue  is  placed  for  10  to  15  minutes  in  a  1  %  solu- 
tion of  acetic  acid,  for  24  hours  in  0.01  %  chromic  acid. 

This  method  is  useful  for  macerating  spinal  ganglia. 

After  washing  well  in  flowing  water  the  teased  ele- 
ments may  be  stained  in  a  1  %  solution  of  methylen- 
blue. 

It  is  best  to  mount  teased  preparations  in  gum  glycerin, 
this  may  be  made  after  the  following  formula,  as  suggested 
by  Farrant. 

Glycerin,  .        .        .        .        .        50  c.  c. 

Water, 50  c.  c. 

Gum  Arabic  (powder),        ...        50  grms. 

Arsenous  acid,  ....          2  grms. 

Or  in  glycerin  gelatin  (Fol.)  : 

Water,  .        .        .        .        .        .  42  c.  c. 

Glycerin,       .        ,        .        .        .        .  38  c.  c. 

Gelatin, 7  grms. 

Carbolic  acid,       .        .        .        .        .          1  grm. 


METHODS  FOR  HARDENING. 


ALCOHOL.     [Strong]. 

When  possible  to  cut  tissue  inlo  small  pieces, 
about  one  quarter  to  one-half  inch  cube,  it  is  best  to 
place  them  at  once  into  95%  alcohol,  here  they 
remain  for  four  to  five  days,  are  then  transferred 
to  absolute  alcohol  for  24  to  48  hours.  It  is  always 
well  to  place  a  layer  of  absorbent  cotton  into  the  harden- 
ing jar  to  prevent  the  tissues  from  resting  on  the  bottom. 
The  fluid  can  then  more  readily  touch  all  surfaces  of  the 
tissue  to  be  hardened.  Gland  tissue,  such  as  salivary 
glands,  and  pancreas,  the  intestinal  canal  are  well  harden- 
ed by  this  method.  Kahlden  states  that  this  method  is 
especially  useful  when  the  tissues  are  to  be  examined  for 
bacteria. 

ALCOHOL.     [Graded]. 

If  it  is  not  possible  or  advisable  to  cut  the  tissue  into 
small  pieces,  it  is  better  to  place  them  in  alcohol  of 
about  60%  in  which  they  remain  for  twelve  hours;  then 
into  alcohol  of  75  per  cent,  for  24  hours  ;  finally  into  alco- 
hol of  95  per  cent,  for  eight  to  sixteen  days,  at  the  end 
of  which  time  they  will  be  ready  for  cutting.  When  used 
in  this  way,  alcohol  penetrates  more  easily  into  the  larsre 
pieces. 

MULLER'S  FLUID.     [Heinrich  Muller]. 

The  "fluid  "  is  made  after  the  following  formula  : 

Potassium  bichromate     .  .  .  .2.5  parts. 

Sodium  sulphate  .'  .  .  .1  part. 

Water         ......      100  parts. 


—  85- 

The  potassium  bichromate  and  sodium  sulphate  are  to 
be  ground  in  a  mortar.  They  dissolve  quickly  if  the  water 
is  heated.  A  large  quantity  of  the,  fluid  may  be  kept  on 
hand  as  it  does  not  deteriorate  by  standing.  Midler's 
fluid  is  especially  to  be  recommended  when  large  pieces 
are  to  be  hardened,  but  it  must  be  remembered  the  hard- 
ening takes  place  very  slowly.  Pieces  of  about  an  inch 
cube  harden  in  two  to  four  weeks,  of  two  inches  cube  1-2 
months  and  larger  masses  proportionately  longer — a 
human  brain  for  instance  needs  to  be  in  the  fluid  from  6  to 
8  months.  Always  use  a  large  quantity  of  the  fluid  and 
change  whenever  it  becomes  turbid.  Weigert  reccftnmends 
that  the  jar  containing  the  tissue  to  be  hardened,  especi- 
ally if  it  be  the  central  nervous  system,  be  kept  in  a  warm 
oven  at  a  temperature  of  30°— i()°C,  a  spinal  cord  may  in 
this  way  be  hardened  in  one  to  two  weeks.  After  hardening' 
in  Miiller's  fluid  thfe  tissues  need  to  be  well  washed  in  flow- 
ing water  for  several  hours,  are  then  placed  into  75  per 
cent,  alcohol  for  two  to  three  days  and  into  95  per  cent, 
for  four  to  six  days ;  they  are  now  ready  for  cutting. 
Hans  Virchow(1)  recommends  that  the  preparation  be  placed 
from  the  Midler's  fluid  into  96  per  cent,  alcohol,  the  tissues 
must  however  be  kept  in  the  dark. 

This  method  is  especially  useful  for  hardening  the 
central  nervous  system,  and  when  necessary  to  harden 
large  masses,  entire  organs,  tumors,  etc.  It  is  not  used  to 
advantage  when  the  finer  structure  of  cells  is  desired. 

BICHLORIDE  OF  MERCURY.     [Lleidenlmin]. 

A ,0.5  per  cent,  sodium  chloride  solution  is  saturated 
with  mercuric  chloride;  to  do  this  bring  seven  to  eight 
grams  of  the  bichloride  of  mercury  into  100  c.  c.  of  the  salt 
solution  and  boil,  the  solution  becomes  supersaturated,  aiid 
on  cooling  some  of  the  bichloride  of  mercury  is  deposited  on 
the  bottom  of  the  bottle  in  the  form  of  needle  shaped  crys- 
tals, and  a  clear  saturated  solution  is  obtained.  This  solu- 


(1)    Quoted  by  Raw  it-/.— Leitftuleu  Ilistologischer  Untevsuchungen. 


-86  — 

tion  does  not  penetrate  well,  so  that  the  pieces  to  be  hard- 
ened must  not  be  larger  than  about  -\  inch  cube,  they 
remain  in  the  fluid  from  two  to  four  hours,  and  are  then, 
either  thoroughly  washed  in  flowing  water  (several  hours) 
and  hardened  in  graded  alcohol,  remaining  in  each  of  the 
solutions  (60  per  cent.—  seventy-five  per  cent. — 95  per  cent, 
alcohol)  for  24  hours  ;  or  they  are  at  once  placed  into  70  per 
cent,  alcohol,  in  which  they  remain  for  24  hours,  changing 
the  alcohol  several  times,  and  (hen  placed  into  95 percent, 
alcohol.  A  few  crystals  of  the  iodide  of  potassium  are 
with  advantage  added  to  the  70  per  cent,  alcohol,  as  bichlo- 
ride of  mercury  is  readily  soluble  in  solutions  of  this  salt. 

It  is  very  necessary  that  the  bichloride  of  mercury  be 
well  washed  out  of  the  tissues,  or  the  student  will  be 
annoyed  with  the  crystals  of  this  salt  making  their  appear- 
ance in  the  sections. 

This  solution  is  one  of  the  best  hardening  fluids  the 
histologist  possesses,  hardens  rapidly  and  well  if  the  pieces 
are  small.  It  may  be  warmly  recommended  for  harden- 
ing small  bits  of  tissue  that  have  been  removed  from 
.growths  for  diagnostic  purposes.  The  mucous  membrane 
of  intestinal  canal,  gland  and  muscle  tissue  are  well  hard- 
ened by  it. 
NITRIC  ACID.  [Benda]. 

Benda  recommends  a  10  per  cent,  solution,  in  this  the 
tissues  remain  from  24  to  48  hours,  are  then  transferred  to 
Mailer's  fluid  for  one  to  two  weeks,  thoroughly  washed  in 
flowing  water  for  several  hours,  the  hardening  is  completed 
in  "graded  alcohol."  This  method  gives  good  results  when 
it  is  desired  to  harden  an  entire  eye,  the  HN03  fixes  the 
tissues  and  it  can  then  be  placed  into  Miiller's  fluid  with- 
out collapsing. 

Skin,  scalp  are  also  well  hardened  after  this  method. 
CHROMIC  ACID. 

The  method  here  given  is  the  one  recommended  by 
Prof.  Glibbes.(1)  A  one-sixth  per  cent,  watery  solution  of 

(1)    Practical  Histology  and  Pathology.    Third  edition,  page  16. 


SCHOOL 


chromic  acid  is  made,  of  this  two  parts  are  mixed  with 
one  part  of  95  per  cent,  alcohol,  the  mixture  is  to  be  well 
stirred.  The  pieces  of  tissue  must  be  small,  about  -J  inch 
cube.  Change  the  fluid  at  the  end  of  the  first,  third  and 
fifth  day;  they  are  hardened  in  eight  to  twelve  days. 
Wash  well  in  flowing  water  for  several  hours,  the  harden- 
ing is  completed  in  "graded  alcohol." 

PICRIC  ACID. 

A  saturated  watery  solution  is  kept  on  hand  ;  filler  be- 
fore using.  The  tissue  needs  to  be  cut  into  small  blocks. 
they  remain  in  the  fluid  for  one  to  three  days,  are  then 
rinsed  in  water,  and  placed  in  80  per  cent,  alcohol,  which 
must  be  changed  as  often  as  it  becomes  yellow  ;  as  soon  as 
none  or  very  little  of  the  acid  is  given  off,  place  in  95 
per  cent,  alcohol.  Peripheral  nerves,  vessels,  elastic 
cartilage,  and  fibro  cartilage  are  well  hardened  in  this 
way.  '  Foetal  bones  are  decalcified. 

FLEMING'S  SOLUTION.     [Chromic,  osmic,  acetic  acid  solution.] 

One  of  the  best  of  hardening  solutions  is  a  mixture  of 
chromic, osmic,  and  acetic  acid  in  the  following  proportions: 

Osmic  acid  (2%  watery  sol.)  ....  4  parts. 
Chromic  acid  (\%  watery  sol.)  ...  15  parts. 

Glacial  acetic  acid       ......        1    part. 

The  solution  may  be  kept  on  hand  in  a  well  stoppered 
bottle.  Tissues  must  be  in  small  pieces,  one  dimension 
of  which,  at  least,  ought  not  to  be  more  than  one- 
twelfth  or  one-eighth  of  an  inch.  The  chromic  acid  might 
penetrate  larger  pieces,  the  osmic  not.  The  tissues  remain 
in  the  solution  about  24  hours,  are  then  thoroughly 
washed  in  flowing  water,  the  hardening 'is  completed  in 
ki  graded  alcohol."  This  solution  is  largely  used  in  hard- 
ening tissues  for  cell  division,  and  for  bringing  out  the 
finer  details  in  the  structure  of  the  protoplasm  and  nucleus. 
Unless  tissues  are  well  washed  it  is  often  hard  to  stain 
them. 


-88- 

HERMANN'S   SOLUTION.     [Platinum  chloride,  osmic,  acetic  acid 
solution.] 

Hermann  uses  in  place  of  the  chromic  acid  in  Fleming's 
solution  a  1  %  watery  solution  of  platinum  chloride,  the 
formula  reading  as  follows: 

Platinum  chloride  (1  %  watery  sol.)    "    .        .        15  parts. 
Osmic  acid  (2%  watery  sol.)  .        .         .      2-4  parts. 

Glacial  acetic  acid  .....          1    part. 

Pieces  need  to  be  small,  they  remain  in  the  solution 
for  24  to  48  hours — washed  in  water — hardened  in  "  graded 
alcohol."  Hermann's  fluid  is  used  with  good  results  in 
hardening  for  karyokinesis,  spermatogenesis,  etc. 

Bichloride  of  mercury  may  with  advantage  be  added 
to  either  Fleming's  or  Hermann's  solution,  it  seemed  to  Dr. 
Bend  a  and  the  author  that  the  accessory  nucleus  was  bet- 
ter fixed  when  this  was  done.  The  following  formulas 
may  be  given: 

Bichloride  of  mercury  (saturated  water  sol.)     .  3  parts. 

Platinum  chloride  (1  %  watery  sol.)          .        .  3  parts. 

Osmic  acid  (1  %  watery  sol.)  .        .         .  3  parts. 

Acetic  acid  (Glacial) 1    part. 

To  be  used  as  Hermann's  fluid. 

Bichloride  of  mercury  (saturated  watery  sol.)  3  parts. 

Chromic  acid  (1  %  watery  sol.)         .        .        .  4  parts. 

Osmic  acid  (2  %  watery  sol.)  ...  2  parts. 

Glacial  acetic  acid  (33  %  watery  sol.)       .        .  1    part. 
To  be  used  as  Fleming's  solution. 


METHODS   FOR   DECALCIFYING  TISSUES. 


Kahlden*  gives  the  following  general  directions  to  be 
observed  when  decalcifying : 

1)  The  tissues  must  first  be  well  hardened  in  alcohol 
or  Miiller's  fluid. 

2)  A  large  quantity  of  the  decalcifying  fluid  needs  to 
be  used,  and  changed  frequently. 

3)  After  decalcification  the  tissues  must  be  thoroughly 
washed  in  flowing  water  for  several  days. 

4)  They  are  again  hardened  in  u  graded  alcohol,"  after 
which  they  are  ready  for  cutting. 

NITRIC  AND  HYDROCHLORIC  ACID. 

Use  the  following  proportions  : 

Nitric  acid  (10  %  watery  solution)  '.        ,        5  parts. 

Hydrochloric  acid  (I  %  watery  solution)          .        5  parts. 

The  decalcification  is  quite  rapid,  the  fluid  needs  to  be 
changed  every  second  or  third  day.  The  tissues  are  from 
time  to  time  taken  from  the  fluid  and  tested,  by  pushing  a 
needle  into  the  bone,  and  if  it  enters  easily  and  without 
grating,  the  decalcification  may  be  considered  complete. 
They  are  then  washed  in  flowing  water  and  hardened  in 
"  graded  alcohol." 

EBNER'S  DECALCIFYING  FLUID. 

The  following  formula  is  taken  from  "  Behren's  Ta- 
bellen": 

"Tfclmik  dcr  Histologisc,hen  Untersuchung  patologlsch-anatomischer 

.    Third  edition.    Page  13. 
10 


-90  — 

Sodium  chloride 2.5  grins. 

Water 100.0  c.c. 

Alcohol .  500.0  c.c. 

Hydrochloric  acid      ......        2.5  c.c. 

This  solution  decalcifies  very  slowly,  but  without  in- 
jury to  the  tissues.  Large  quantities  (10  to  20  times  the 
bulk  of  the  tissue)  need  to  be  used,  arid  one  or  two  c.c.  of 
hydrochloric  acid  are  daily  added  to  the  fluid  until  decal- 
ciiication  is  complete.  Wash  very  thoroughly  in  flowing 
water,  and  harden  in  li  graded  alcohol.'' 

HAUG'S  CHROM-OSMIUM  SOLUTION. 

Osmic  acid  (1  %  watery  solution)        .         .         .  10  c.c. 

Chromic  acid  (1  %  watery  solution)             .        .  25  c.c. 

Water fiO  c.c. 

Useful  for  decalcifying  cochlea;  acts  very  slowly. 
Wash  in  flowing  water,  and  harden  in  alcohol. 

LEPKOWSKI'S  METHOD.* 

Small  pieces  of  tooth  or  bone  are  placed  in  the  follow- 
ing solution  : 

Chloride  of  gold  (1  %  watery  solution)     .        .        6  parts. 
Formic  acid          ....       Yv    .  .        .        3  parts. 

Tissues  are  decalcified  in  two  or  three  days.  The 
chloride  of  gold  is  deposited  in  the  dentinal  tubules  or 
canaliculi,  so  that  the  tissue  will  prove  to  be  stained  and 
decalcified  at  the  same  time. 

*Anatomischer  Anzeiger,  1892. 


IMPREGNATION  OF  TISSUES. 


SILVER   NITRATE. 

Used  for  staining  the  endothelial  membranes  lining 
blood-vessels  and  serous  cavities.  An  albumenate  of  silver 
is  formed  with  the  intercellular  cement  between  the  endo- 
thelial cells;  the  silver  is  reduced  on  exposing  to  sun- 
light. A  1.0  per  cent,  watery  solution  of  the  silver  nitrate 
is  used.  To  make  a  preparation  of  an  endothelial  mem- 
brane, the  peritoneum  of  a  frog  may  be  selected  ;  the  fol- 
lowing steps  are  taken:  make  a  small  opening  through 
the  abdominal  wall  of  a  frog  near  the  sternum,  inject  10 
to  20  c.  c.  of  the  one  per  cent,  solution  of  silver  nitrate; 
while  injecting  the  abdomen  is  gently  kneaded,  so  that  the 
fluid  may  be  well  distributed  over  the  abdominal  cavity. 
In  15  to  20  minutes  the  abdomen  is  opened,  the  intestinal 
canal  with  the  mesentery  is  removed,  the  latter  (without 
removing  from  the  intestines)  is  spread  out  on  a  "cork 
board,"  is  now  immersed,  preparation  side  up,  in  80  per 
cent,  alcohol  (it  will  be  necessary  to  fix  small  lead  weights 
to  the  cork  to  bring  it  under  the  alcohol)  and  placed  in 
the  sunlight;  as  soon  as  the  tissue  shows  a  brown  color  it 
is  ready  for  study.  It  may  be  mounted  in  gum  glycerin 

or  balsam. 

GOLD  CHLORIDE  METHOD. 

After  Golgi. 

Small  pieces  of  fresh  tissue  are  placed  into  a  0.5 
per  cent,  solution  of  arseneous  acid,  in  which  they  remain 
until  they  become  transparent  (usually  five  to  ten  min- 
utes), they  are  then  placed  into  a  0.5  per  cent,  watery  sol. 
of  gold  chloride  about  half  an  hour,  in  the  dark.  They  are 
now  pl;u-e<l  in  a  one  per  cent,  solution  of  arseneous  acid 


in  which  they  are  exposed  to  sunlight  until  the  gold  is 
reduced.  Mount  in  gum-glycerin.  Used  for  staining 
axis  cylinders  and  nerve  end  organs,  but  especially  for 
muscle  end  plates. 

After  Hanvier. 

For  staining  nerve  endings  in  epithelial  tissue,  cornea, 
etc ,  place  a  small  piece  of  the  tissue  into  the  following 
solution  : 

Chloride  of  gold  (1  per  cent,  watery  sol.),          .       4  parts. 
Formic  acid,          .         .        .        .        .        .        .1  part. 

In  this  they  remain  for  twenty  four  hours,  in  the  dark. 
Expose  to  sunlight,  in  distilled  water,  to  which  a  few 
drops  of  acetic  acid  have  been  added,  until  the  gold  is 
reduced. 

SILVER  NITRATE   AND  BICHROMATE  OF  POTASH   METHOD. 

[Golgi,  Ramon  J.  Cajal  and  Lenhossek]. 

Used  for  staining  nerve  cells  and  their  processes  in 
the  central  neryous  system  and  the  periphery.  The  for- 
mulas here  given  are  taken  from  Lenhossek's  u  referat"  in 
fortsckritte  der  Medicin,  August  and  September,  1892. 

Golgi'' s  Slow  Method. 

Small  pieces  of  brain  or  spinal  cord  are  hardened  in  a 
2  per  cent,  solution  of  bichromate  of  potassium  from  twenty 
to  thirty  days ;  are  then,  without  washing,  transferred  to 
a  0.5  per  cent,  solution  of  silver  nitrate,  in  which  they 
remain  from  twenty-four  to  forty-eight  hours ;  or  in  place  of 
the  silver,  a  0.5  per  cent,  solution  of  bichloride  of  mercury 
may  be  used,  in  this  they  remain  from  two  to  four 
weeks. 

The  Mixed  Golgi  Method. 

The  tissues  to  be  hardened  and  stained  are  placed 
in  a  large  quantity  (20  to  30  times  the  bulk  of  the  tissues 
used),  of  the  following  solution  : 

Bichromate  of  potassium  (1  per  cent,  watery  sol.),  8  parts. 
Osmic  acid  .  .  (1  per  cent,  watery  sol.),  1  part. 


—  93  — 

In  this  this  they  remain  four  or  five  days ;  are  then 
transferred  to  0.75  per  cent,  solution  of  silver  nitrate 
twenty-four  to  thirty  hours. 

Rapid  Method.     [Ramon  J.  Cajal]. 

The  following  solution  is  now  largely  used: 
Bichromate  of  potassium  (3  to  5  per  cent,  sol.),       4  parts. 
Osmic  acid,        .        .        (1  per  cent,  watery  sol  ),  1  part. 

If  it  is  desired  to  stain  neuroglia  cells,  allow  the  tis- 
sues to  remain  in  the  solution  two  or  three  days,  if 
nerve  cells,  three  to  five  days;  if  nerve  fibres  and 
collateral  branches,  five  to  seven  days.  They  are  then 
transferred  to  a  one  per  cent,  watery  solution  of  silver 
nitrate,  in  which  they  remain  from  twenty  four  to  thirty- 
six  hours.  Lenhossek  adds  one  drop  of  formic  acid  to  200 
c.  c.  of  the  silver  solution. 

These  methods  give  the  best  results  when  embryonic 
tissues  or  tissues  taken  from  newly  born  or  young  animals 
are  employed. 

Often  when  the  methods  above  noted  do  not  give  good 
results  at  the  first  trial,  they  may  with  success  be  repealed 
on  the  same  tissue.  If  after  the  tissues  have  been  two  or 
three  days  in  the  silver  nitrate,  the  trial  sections  (free- 
hand sections  cut  from  the  blocks,  mounted  and  examined 
in  95  per  cent,  alcohol)  show  no  staining,  they  may  again 
be  placed  in  the  bichromate  of  potassium  solution  for 
several  days,  and  then  again  into  silver.  Good  results  fol- 
low a  second  and  even  the  third  trial. 

Sections  are  cut  into  95  per  cent,  alcohol;  they  may 
be  mado  either  "free-hand  "  or  after  embedding  in  celloi- 
din,  with  the  microtome.  From  the  alcohol,  sections  are 
placed  for  1.5  minutes  into  creosote,  then  washed  from  three 
to  five  minutes  in  the  oil  of  turpentine,  out  of  which  they 
are  takon  and  arranged  on  the  slide.  The  excess  of  oil 
of  turpentine  is  removed  with  filter  paper,  and  sections 
are  covered  with  balsam.  The  slide  is  now  carefully 
heated  over  an  alcohol  ilame,  until  the  balsam  becomes  so 


—  94- 

thick  that  on  cooling,  it  at  once  hardens  (three  to  live  min- 
utes of  careful  heating;  are  required,  allow  no  bubbles  to 
form);  while  the  balsam  is  yet  warm  cover  with  a  cover- 
glass  which  has  been  passed  through  the  flame  several 
times  ;  after  cooling  the  preparation  is  ready  for  study.  It 
will  be  found  when  mounted  in  this  way  Golgi  preparations 
do  not  fade. 


METHODS  FOR  EMBEDDING  AND  CUTTING 
SECTIONS. 


Free-hand  Gutting.  It  is  usually  necessary  to  embrace 
the  tissues  to  be  cut  with  some  material,  which  gives  no 
resistance  to,  and  does  not  injure  the  knife,  at  the  same 
time  is  firm  enough  to  give  support.  Pathologists  have  for 
a  long  time  used  small  pieces  of  amyloid  liver  which  had 
been  hardened  in  alcohol  or  Mailer's  fluid.  If  these  can 
not  be  obtained,  pig's  or  calf's  liver  will  answer.  Elder- 
pith  is  also  used.  The  little  blocks  of  liver  tissue,  or  the 
elder  pith  rods,  are  divided  into  two  parts,  between  which 
the  tissue  to  be  cut  is  embraced.  It  can  now  be  firmly 
held  between  the  thumb  and  the  index  finger  of  the  left 
hand.  A  razor,  which  is  flat  on  one  side  (the  under  while 
in  use),  is  employed.  Its  .upper  surface  is  well  covered 
with  80  per  cent,  alcohol.  Try  to  make  the  section  with 
one  continuous  cut,  resting  the  blade  of  the  .knife  on  the 
index  finger.  Sections  which  answer  for  purposes  of  orien- 
tation can  easily  be  made  in  this  way.  They  are,  however, 
not  to  be  compared  with  the  ones  that  can  be  cut  with  the 
microtome  when  the  tissues  are  properly  embedded. 

A  great  many  methods  for  embedding  tissues  are  in 
use,  the  principal  involved  is  in  all  the  same.  In  the 
one  case  the  tissues  are  permeated  with  substances  that 
are  fluid  when  warm  and  become  hard  enough  to  cut  on 
cooling;  others  need  to  be  fro/en  to  give  them  this  con- 
sistency ;  in  others  again,  the  embedding  mass  hardens,  on 
the  evaporation  of  a  solvent  which  was  used  to  bring  it  to 
a  fluid  state.  The  methods  for  embedding  in  celloidin,  \\\ 


—  96  — 

paraffin,  and  in  a  solution  of  gum  arable  (for  the  freezing 
microtome)  are  here  given.  It  is  deemed  beyond  the  scope 
of  these  notes  to  go  into  the  methods  for  cutting  sections, 
they  will  be  taught  in  the  laboratory. 

EMBEDDING  FOR  CUTTING  ON  THE  FREEZING  MICROTOME. 

To  prepare  the  tissues  for  cutting  on  the  freezing 
microtome,  the  following  steps  are  taken:  remove  the 
alcohol  from  the  hardened  tissues  by  allowing  them  to 
remain  in  water  about  eight  hours,  they  are  then  trans- 
ferred to  a  solution  of  gum  arabic,  (solution  should  be 
about  as  thick  as  syrup,  and  is  made  by  dissolving  the 
gum  arabic  in  hot  water,  and  straining  through  a  cloth; 
allow  to  cool  before  using.)  in  about  six  hours  they  are  per- 
meated with  the  gum  arabic  and  are  ready  for  freezing. 
Sections  need  to  be  cut  into  warm  distilled  water,  this 
removes  the  gum  arabic.  Before  staining  or  placing  in 
alcohol  they  must  again  be  washed  in  distilled  water. 

CELLOIDIN  OR  COLLODIUM.* 

A  stock  solution  of  celloidin  or  collodium  is  kept  on 
hand.  This  is  made  by  adding  celloidin  or  collodium  to 
a  mixture  of  equal  parts  of  absolute  alcohol  and  ether 
until  a  thick  solution  is  obtained.  The  stock  solution 
needs  to  be  kept  in  a  u  well  stoppered"  bottle,  as  it  thick- 
ens on  the  evaporation  of  the  alcohol  and  ether.  The  steps 
for  embedding  tissues  are  the  following: 

I.  From  the  95  per  cent,  alcohol  the  tissues  are  placed 
into  absolute  for  2-4  to  48  hours. 

II.  Into  a  mixture  of  equal  parts  of  absolute  alcohol 
and  ether  from  one  to  two  days. 

III.  Into  a  thin  solution  of  celloidin,, consisting  of 
one  part  of  the  "stock  solution"  and  two  parts  of  the 

*The  methods  for  embedding  in  celloidin  and  collodium  are  the  same, 
the  results  seem  to  the  author  equally  good;  collodium  is  cheaper.  The  steps 
here  given  for  the  one  will  answer  for  the  other. 


—  97  — 

alcohol  and  ether  mixture  (equal  parts) ;  in  this  they 
remain,  according  to  the  size  ot  the  pieces  embedded,  from 
two  or  three  days  to  as  many  weeks. 

IV.  Into  the  "  stock  solution  "  for  an  equal  length  of 
time.  The  tissues  are  ready  for  further  treatment  as  soon 
as  they  are  thoroughly  permeated  with  the  celloidin;  no 
definite  time  can  be  fixed  for  this.  Loose  tissues,  such  as 
lung,  are  permeated  in  three  or  four  days;  skin  in  two 
or  three  weeks;  brain  or  spinal  cord,  especially  if  the 
pieces  are  large,  in  three  to  six  weeks. 

As  soon  as  permeation  seems  complete,  some  of  the 
u  stock  solution  "  is  poured  into  a  flat  glass  dish  (enough 
to  cover  well  the  tissues  to  be  embedded),  into  this  the  tis- 
sues are  brought,  and  if  several  pieces  are  to  be  embedded, 
they  are  to  be  so  arranged  that  a  small  area  of  celloidin 
(one fourth  of  an  inch  wide)  surrounds  each  piece.  The 
dish  is  now  covered  with  a  glass  plate  and  placed  under  a 
bell-jar.  The  alcohol  and  ether  evaporate  very  slowly,  on 
doing  so  the  celloidin  hardens.  In  case  the  glass  plate  fits 
tightly,  push  it  to  one  side,  thus  leaving  a  small  space 
uncovered.  Several  days  are  required  for  the  celloidin  to 
become  hard.  As  soon  as  the  mass  is  so  firm  that  in  can  not 
be  indented  with  the  finger,  it  is  removed  from  the  glass 
dish,  and  can  now  be  "  trimmed  "  into  a  square  block  con- 
taining the  tissue;  or  into  several,  if  a  number  of  pieces 
were  embedded.  These  celloidin  blocks  are  now  to  be  fixed 
to  small  cylinders  of  hard  wood.  The  diameter  of  the 
wooden  blocks  should  be  a  little  larger  than  the  celloidin 
block,  and  about  one-half  an  inch  long.  This  is  done  by 
immersing  one  end  of  the  wooden  cylinder  in  the  "  stock 
solution,''  a  layer  of  celloidin  is  in  this  way  spread  over 
this  end;, the  block  of  celloidin  is  now  pressed  against  this 
layer  of  celloidin,,  placed  under  a  bell-jnr  for  about  an  hour, 
and  it  will  be  found  to  adhere  quite  firmly. 

On  cutting  sections  of  a  tissue  embedded  in  celloidin, 
the  knife  should  be  moistened  with  80  percent,  alcohol; 
sections  are  cut  into  weak  alcohol  or  distilled  water.  The 


—  08  — 

celloidin  blocks,  even  after  they  are  fixed  to  the  wooden  cyl- 
inders, may  be  kept  for  a  long  time  in  TO  per  cent,  alcohol. 
The  cellorlin  method  is  now  very  largely  used;  is 
especially  useful  when  desired  to  cut  sections  of  the  cen- 
tral nervous  system  ;  of  an  entire  eye  ;  of  tissues  or  organs 
containing  much  fibrous  tissue.  It  may  be  used  whenever 
not  very  thin  sections  (less  than  10  //.  to  15  //.)  are  re- 
quired. The  celloidin  or  collodium  need  not  be  removed 
before  staining  or  mounting;  and  unless  the  aniline  dyes 
are  used  for  coloring,  very  little  stain  is  taken  by  the  cel- 
loidin. The  sections  need  to  be  cleared  in  oil  of  bergamot, 
as  the  oil  of  cloves  dissolves  the  celloiddn.  The  following 
method  for  staining  and  mounting  celloidin  sections  in 
series,  is  recommended  by  Weigert :  * 

I.  A  clean  glass  plate  is  covered  with  a  thin  layer  of 
a  solution  of  collodium ;  this  is  to  be  spread  out  as  evenly 
as  possible  over  the  entire  surface.    The  plate  is  now  placed 
on  edge,  the  collodium  allowed  to  dry;  care  being  taken 
to  keep  the  dust  from  it. 

II.  A  section  is  placed  on  a  strip  of  "closet  paper," 
near  one  end.      Succeeding  sections,  as  soon  as  cut,  are 
placed  to  the  right  of  it.     They  are   removed  from   the 
knife  to  the  strip  of  paper,  by  holding  the  paper  extended 
under  the  knife,  and  slipping  the  section  onto  it.    To  keep 
the  sections  from  drying,  the  strip  of  paper  is,  after  the 
removal  of  every  section,  placed  in  a  flat  dish,  in  which 
several  layers  of  filter  paper,  have  been  spread  out  and 
thoroughly  saturated  with  80  per  cent,  alcohol.     On  each 
strip  of  paper  is  arranged  only  one  row  of  sections ;  the 
strips  are  kept  in  the  dish  in  the  order  used. 

III.  As  soon  as  a  number  of  strips  have  been  cov- 
ered with  sections,  they  are  arranged,  sections  downward, 
on  the  layer  of  collodium    above   described,  and  gently 
pressed  to  it.    The  strips  of  the  paper  can  now  be  removed, 
the  sections  adhering  to  the  layer  of  collodium.     Several 

*  Taken  from  Rawitz   Leitfaden  der  histologischen  Untersuchungen. 
Page  37. 


—  99  — 

layers  of  filter  paper  are  now  pressed  over  the  sections,  in 
this  way  removing  as  much  of  the  alcohol  as  possible. 

IV.  Before  the  sections  have  time  to  dry  a  layer  of 
the  collodium  solution  is  poured  over  them,  equally  dis- 
tributed, and  allowed  to  dry.  As  soon  as  dry  the  plate 
may  be  placed  into  80  per  cent,  alcohol,  where  it  may  be 
kept,  or  into  the  stain,  the  layer  of  collodium  (containing 
the  sections)  then  separates  from  the  glass  plate,  and  can 
be  treated  as  a  single  section. 

PARAFFIN  EMBEDDING. 

The  tissues  are  permeated  with  melted  paraffin ;  on 
cooling,  the  paraffin  congeals  and  can  then  be  cut.  The 
steps  for  embedding  are  the  following: 

1).  From  95  per  cent,  alcohol  the  tissues  are  trans- 
ferred to  absolute,  here  they  remain  from  12  to  24  hours. 

2).  They  are  then  placed  into  a  substance  which  at 
the  same  time  mixes  freely  with  absolute  alcohol  and  is  a 
solvent  for  paraffin;  turpentine,  choloroform,  oil  of  origa- 
num, and  many  other  substances  are  in  use,  the  tissues 
remaining  in  any  one  of  the  substances  named  from  two 
to  six  hours. 

3).  They  are  then  placed  in  melted  soft  paraffin  in 
which  they  remain  from  one  to  three  hours.  The  paraffin 
known  as  "  soft"  has  a  melting  point  of  about  43°  to  45°  C. 
It  is  kept  in  a  fluid  state  in  a  water  bath,  the  temperature 
of  which  can  be  regulated;  it  should  not  exceed  52°  C. 

4).  From  the  "soft  paraffin,"  the  tissues  are  trans- 
ferred to  melted  hard  paraffin*  this  is  usually  a  mixture 
of  equal  parts  of  "soft"  and  a  paraffin  with  a  melting 
point  of  55°  0.,  and  answers  very  well  for  ordinary  room 
or  laboratory  temperature.  During  the  summer  months, 
it  may  be  necessary  to  use  two  parts  of  the  paraffin  with 
55°  C.  melting  point,  and  only  one  of  the  "soft,"  and  in 
very  warm  weather  even  less  of  the  "soft."  In  the  hard 
paraffin,  the  tissues  remain  from  two  to  six  hours.  It  is 
essential  that  the  paraffin  while  in  use  for  embedding 


—  100  — 

should  be  at  a  constant  temperature;  this  can  easily  he 
done  when  a  water  bath  with  temperature  regulator,  such 
as  is  found  in  most  laboratories,  is  at  hand.  A  very  simple 
apparatus,  and  one  that  meets  the  requirements  quite  well, 
is  shown  in  Fig.  I.  It  consists  of  a  tripou  (F);  a  copper 


B. 


A.         'Or 


Fig.  1.  Simple  paraffin  bath;  (A)  copper  plate;  (B)  paraffin  tray;  (C) 
loop  of  filter  paper;  (D)  area  of  melted  paraffin ;  (.E)  area  of  unmelted  paraffin ; 
(F)  tripod;  (G)  plane. 

plate  (A)  about  fifteen  inches  long,  five  inches  wide  and 
one-eighth  to  three-sixteenths  of  an  inch  thick;  two  tin 
trays  (B)  (only  one  is  shown  in  the  diagram),  these  are 
ten  inches  long,  two  wide,  and  three  deep.  They  are 
partly  filled  with  paraffin;  the  one  with  soft,  the  other 
with  the  hard.  If  an  alcohol  lamp  or  a  Bunsen  burner  be 
so  placed  that  the  end  of  the  flame  (G)  touches  one  end  of 
the  copper  plate,  as  shown  in  diagram,  and  the  trays  con- 
taining the  hard  and  soft  paraffin  be  placed  on  the  copper 
plate  toward  the  end  away  from  the  flame,  it  will  be  found 
that  after  a  short  time,  fifteen  to  thirty  minutes,  the  par- 
affin in  the  end  of  the  trays  near  the  flame  will  be  melted 
while  in  the  other  end  it  is  yet  hard,  by  reason  of  the  fact 
that  the  flame  end  of  the  copper  plate  has  a  higher  tempera- 
ture than  the  opposite  end.  With  a  little  patience  the 
trays  may  be  so  adjusted,  by  moving  them  toward  or  away 
from  the  flame,  that  about  half  of  the  paraffin  will  be 
melted,  the  rest  not.  As  shown  in  diagram  (D)  represents 


—  101  — 

the  area  of  melted  paraffin,  (E)  of  the  unmelted.  It  of 
course  stands  to  reason,  that,  if  the  two  trays  be  placed 
side  by  side  on  the  copper  plate,  the  one  containing  the 
hard  paraffin  needs  to  be  nearer  the  flame  than  the  one 
with  the  soft,  to  obtain  in  each  an  area  of  melted  and  un- 
rnelted  paraffin.  In  either  tray,  the  area  of  unmelted 
paraffin  acts  as  a  thermometer,  the  adjoining  melted  par- 
affin must  have  a  temperature,  which  when  expressed  in 
degrees,  is  about  the  melting  point  of  the  paraffin  in  ques- 
tion ;  about  45°  0.  for  the  soft,  50°  0.  for  the  hard.  It  is 
not  advisable  to  allow  the  tissues  to  rest  on  the  bottom  of 
the  tray.  A  loop  can  easily  be  made  with  a  strip  of 
filter  paper  about  two  inches  wide,  this  is  supported  from  a 
wire  or  glass  rod,  and  allowed  to  hang  in  the  area  of  melted, 
near  the  edge  of  the  unmelted  paraffin,  (C)  in  the  figure. 
The  tissues  are  placed  on  the  filter  paper.  When  the 
tissues  are  thoroughly  permeated  with  the  hard  paraffin,  a 
rectangular  trough  is  made  with  two  metalic  L's,  rest- 
ing on  a  glass  plate,  and  filled  with  hard  paraffin.  Into 
this  the  tissue  is  placed,  by  means  of  a  pair  of  small  forceps, 
which  before  using  are  warmed  over  an  alcohol  flame. 
The  piece  of  tissue  is  placed  in  one  end,  and  so  arranged, 
that  the  plane  in  which  it  is  to  be  cut,  is  at  right  angles  to 
Hie  long  axis  of  the  trough.  The  paraffin  is  now  allowed 
to  cool,  and  as  soon  as  a  film  forms  over  it,  the  trough  is 
placed  in  cold  water;  :his  quickly  congeals  the  paraffin. 
The  metalic  L's  are  removed;  the  paraffin  block  can  now 
be  taken  from  the  glass  plate  and  is  ready  for  cutting. 
The  knife  used  for  cutting  paraffin  sections  must  be  dry, 
and  if  perfectly  embedded  very  thin  sections  can  be  cut, 
thinner  than  when  embedded  after  any  other  method. 

Before  mounting  or  staining  a  paraffin  sections,"  they 
must  be  fixed  to  the  slide  or  cover  glass.  To  do  this,  one 
of  the  following  methods  may  be  used: 

Albumen  Fixative.  (Mayer.)  The  u  albumen  fixa- 
tive'7 consists  of  i-qual  parts  of  white  of  egg  and  glycerin. 
Jt  is  prepared  by  chopping  the  white  of  an  egg  with  a  pair 


102 


of  scissors,  then  straining  it  through  muslin  or  linen ;  it  is 
now  mixed  with  an  equal  quantity  of  glycerin.  The  glycerin 
and  white  of  egg  are  to  be  thoroughly  mixed  by  stirring 
with  a  glass  rod. 

A  small  drop  of  the  albumen  fixative  is  placed  on  a 
slide,  and  spread  in  a  veyy  thin  layer  with  a  clean  glass  rod, 
or  with  a  dry  and  clean  linger.  The  section  is  now  placed 
on  the  albumen  fixative,  and  pressed  to  the  slide.  If  the 
section  is  stained,  the  paraffin  is  melted  by  holding  the 
slide  or  cover-glass  over  an  alcohol  flame,  or  by  placing 
them  on  a  water-bath,  until  the  paraffin  begins  to  melt; 
then  cover  with  a  few  drops  of  oil  of  turpentine,  this  dis- 
solves the  paraffin  ;  remove  excess  of  oil  with  filter  paper 
and  mount  in  balsam.  If  it  is  desired  to  stain  the  sec- 
tions after  they  are  fixed  to  the  slide  or  cover-glass,  the  fol- 
lowing steps  are  taken  to  remove  the  paraffin  : 

1.  Heat  quickly  over  the  flame  until  the  paraffin  be- 
gins to  melt. 

2.  Place    the    slide  or    cover-glass   into   turpentine, 
(chloroform  or  toluol  may  also  be  used)  until  the  paraffin 
is  dissolved. 

3.  Transfer  to  absolute  alcohol  for  three  to  five  min- 
utes— this  removes  the  turpentine. 

4.  Transfer  to  95%  alcohol  for  five  minutes. 

5.  Transfer  to  70%  alcohol  for  several  minutes. 

6.  Remove  alcohol  by  placing  slide  or  cover  into  dis- 
tilled water.     Sections  can  now  be  stained. 

It  is  often  quite  difficult  to  remove  folds  from  paraffin 
sections,  and  especially  if  sections  are  large,  before  fixing 
to  the  slide  or  cover-glass  with  "albumen  fixative;'1  the 
author  has  used  with  success  this  simple  method  : 

An  evaporating  dish  is  partly  filled  with  distilled 
water;  on  this  the  sections  are  placed.  The  water  is  now 
slowly  heated  by  holding  the  evaporating  dish  over  an 
alcohol  flame.  It  will  be  noticed  that  as  the  temperature 
of  the  water  is  elevated,  and  the  paraffin  in  and  about  the 
sections  begins  to  soften,  the  sections  spread  out  over  the 


—  103- 

surface  of  the  water.  The  water  is  heated  until  all  folds 
are  obliterated.  Care  should  be  taken  not  to  get  the  water 
hot,  enough  to  melt  the  paraffin.  A  slide  or  cover-glass,  on 
which  a  thin  layer  of  the  albumen  fixative  is  spread,  is 
now  passed  under  one  of  the  sections;  on  it  the  section  is 
caught  and  withdrawn  from  the  water.  Allow  all  the 
water  to  evaporate,  this  usually  taking  8  to  12  hours,  at 
the  end  of  which  time  the  .paraffin  may  be  removed  as  above 
described.  This  method  is  very  useful  for  mounting 
u  serial  sections." 

GauVx  XO  per  refit.  Alcohol  Method.  A  few  drops 
of  50  per  cent,  alcohol  are  placed  on  a  slide  or  cover- 
glass;  on  this  the  sections  are  placed.  As  the  alco- 
hol evaporates,  the  sections  are  fixed  to  the  slide  or  cover; 
12  to  24  hours  are  required,  and  it  is  best  to  place  them  in 
a  warm  oven  at  a  temperature  of  40°  C.  The  paraffin 
is  removed  in  the  same  manner  as  when  sections  are 
fixed  with  albumen  fixative.  The  slide  or  cover  must 
be  very  clean,  even  then  the  sections  are  o'ten  loosened 
while  the  paraffin  is  being  removed.  Sections  from  alco- 
hol or  sublimate  hardened  tissues  seem  to  be  most  firmly 
fixed. 


METHODS  FOR  INJECTING. 


When  it  is  desired  to  bring  to  prominence  the  relation 
between  the  blood  vessels  and  the  other  elements  of  a 
tissue  or  an  organ,  it  is  necessary  to  inject  the  vessel. 
This  is  best  done  by  means  of  substances  which  are  fluid 
when  warm,  but  harden  on  cooling.  Gelatin,  which  has 
been  colored  with  some  dye,  is  usually  employed.  The 
directions  for  making  two  such  injecting  masses  are  here 
given.  Much  experience  is  required  to  inject  successfully  ; 
too  much  space  would  be  required  to  go  fully  into  the 
methods  used,  they  will  be  demonstrated  in  the  course  on 
methods. 

C1RMIN  GELATIN.     (Gerlach.) 

The  formula  is  taken  from  Behren's  Tabellen. 

Oarmin,  lOgrms.) 

Ammonium  hydrate,       .         .         .         .  1  c.  c.    >-  I. 

Water,  '.; .          8  c.  c.    ) 

Gelatin,  12  grms.)    TT 

Water, 16  grms.f  J 

The  gelatin  is  cut  into  fine  pieces,  placed  in  an 
evaporating  dish,  the  water  is  then  added;  in  this  the 
pieces  soak  about  12  hours.  The  gelatin  is  then  dis- 
solved over  a  water-bath.  To  solution  II,  add  solution  I, 
which  is  prepared  by  dissolving  the  carmin  in  the  ammo- 
nium hydrate  and  water,  over  a  water-bath;  add  slowly, 
while  constantly. stirring.  The  mass  is  now  alkaline,  and 
unless  neutralized  would  stain  the  bloodvessels  and  sur- 
rounding tissue.  The  neutralization  is  accomplished  by 
means  of  glacial  acetic  acid,  which  is  added  drop  by  drop 
until  no  ammonia  is  detected  by  the  sense  of  smell ;  stir- 


—  105  — 

ring  well  after  every  drop.  It  will  also  be  noticed  that 
the  mass  changes  its  color,  becoming  a  brighter  red.  If 
the  mass  becomes  too  acid'it  appears  granular  if  a  drop  is 
examined  under  the  microscope ;  it  is  in  this  state  not 
useless,  but  the  resulting  injection  is  never  so  good,  as 
when  the  mass  is  neutral. 

Before  using,  the  mass  must  be  strained  through  a 
piece  of  flannel  which  has  been  dipped  in  hot  water. 
The  canula,  syringe,  and  animal  must  be  kept  warm  dur- 
ing the  injection.  If  the  entire  animal  is  to  be  injected, 
the  canula  is  to  be  tied  into  the  arch  of  the  aorta,  through 
the  left  ventricle ;  If  a  single  organ,  through  its  main 
artery. 

BERLIN  BLUE  MASS.     (Harting,  as  given  by  Rawitz.) 

One  part  of  oxalic  acid  is  "  rubbed  up  "  in  a  glass 
mortar ;  tojkthis  is  added  one  part  of  Berlin  blue,  and 
while  constantly  stirring,  12  parts  of  water.  To  an  equal 
quantity  of  warm**gelatin  solution  (made  as  above 
directed),  add  the  Berlin  blue  solution,  slowly  and  while 
stirring.  Filter  through  a  piece  of  flannel  before  using. 


METHODS  FOR  STAINING. 


It  has  long  been  known  that,  when  properly  hardened 
tissues  are  subjected  to  the  action  of  coloring  matters, 
certain  elements  of  the  tissues,  even  certain  parts  of  the 
cell,  show  greater  affinity  for  the  stain  than  others.  This 
selective  action  noticed  in  so  many  stains,  warrants  the 
place  they  hold  in  histological  technic.  Of  the  great  num- 
ber in  use,  a  few  of  the  most  trustworthy,  and  such  as  can 
be  most  easily  made  and  used,  are  here  given. 

HAEMATOXYLIN  SOLUTIONS. 

Bcehmer^s  solution. — This  solution  is  one  of  the  best 
in  use,  and  is  made  after  the  following  formula  : 

Haematoxylin  crystals        ....     1  grm.)       i     T 
Absolute  alcohol  .         10  c.  cm.j   k 

Potash  alum        .         .        .     •  •'..'      .         10  grms.)       i  ]T 
Distilled  water  200c.cm.f  l 

The  crystals  of  haematoxylin  are  dissolved  in  the 
absolute  alcohol  and  kept  in  a  well  stoppered  bottle  for  24 
ho urs  (solution  I). 

The  alum  is  dissolved  in  warm  distilled  water,  allow 
to  cool,  keeping  it  free  from  dust  (solution  II).  Add 
solution  one  to  solution  two,  stir  and  keep  in  an  open  dish 
for  about  a  week  ;  filter  and  the  solution  is  ready  for  use. 

The  tissues  need  to  be  stained  in  section,  the  steps  are 
as  follows : 

1.  The  sections  come  from  distilled  water  into  the 
stain,  in  which  they  remain  from  five  to  ten  minutes. 

2.  Transfer  to  a  0.5  per  cent,  potash  alum   solution 
for  five  minutes. 


-.107-- 

3.  Wash  in  distilled  water — dehydrate  in  alcohol — 
clear  in  oil  of  cloves  or  oil  of  bergamot — mount  in  bal- 
sam. 

If  desired,  the  stain  may  be  diluted  15  to  20  times  with 
a  0.5  per  cent,  potash  alum  solution,  in  this  diluted  stain 
sections  remain  12  to  24  hours,  are  then  treated  as 
above. 

If  the  sections  are  overstained,  they  may  be  decolor- 
ized in  a  one  per  cenl.  solution  of  acetic  acid  until  the 
proper  tone  is  obtained;  they  must  however,  be  well  washed 
in  water  after  such  decolorization. 

Ehrlictts  hcematoxylin  solution. — This  solution  can  be 
kept  for  a  lon.g  time,  it  seems  to  improve  with  age. 

Hrematoxylin  crystals        ....       V  2grms. 

Absolute  alcohol         ......  20  c.  cm. 

Glycerin .        .  100  c.  cm. 

Distilled  water 100  c.  cm. 

Absolute  alcohol 80  c.  cm. 

Glacial  acetic  acid 10  c.  cm. 

Potash  alum  to  saturation. 

Mix  the  distilled  water,  the  glycerin,  the  80  c.  cm.  of 
absolute  alcohol'  and  the  10  c.  cm.  of  glacial  acetic  acid. 
Dissolve  the  hgematoxylin  crystals  in  the  20  c.  cm.  of  abso- 
lute alcohol,  and  add  to  the  above  solution,  and  shake 
well  for  several  minutes. 

The  solution  so  obtained  should  have  a  reddish  color 
and  is  now  to  be  saturated  with  the  alum  ;  filter  at  the  end 
of  24  hours. 

Ehrlich's  haematoxylin  needs  to  mature  from  one  to 
two  months  before  it  can  be  used.  Steps  for  staining  are 
as  follows  : 

1.  Sections  remain  in  the  stain  from  10  to  30  minutes 
(they  do  not  overstain). 

2.  Wash  in  distilled  water — dehydrate — clear  in  oil 
of  cloves  or  bergamot — mount  in  balsam. 

Delafieltfs  hciematoxylin  solution. — The  formula  is 
taken  from  Behren's  Tabellen : 


—  108  — 

HcTematoxylin  crystals 4  grins. 

Absolute  alcohol         .        .        .         .        .  25  c.  cm. 

Ammonium  alum 52  grins. 

Distilled  water             400  c.  cm. 

Glycerin .  100  c.  cm. 

Methyl  alcohol 100  c.  cm. 

The  hgeinatoxylin  crystals  are  dissolved  in  the  absolute 
alcohol,  the  alum  in  the  hot  water;  as  soon  as  the  alum 
solution  cools  add  the  haematoxylin  solution.  Allow  to 
stand  in  a  wide  vessel  from  three  to  four  days,  filter  and 
add  ihe  glycerin  and  methyl  alcohol. 

This  solution  is  very  useful  for  staining  tissue  in  mass, 
before  using  dilute  three  to  five  times  with  distilled 
water. 

The  pieces  of  tissue,  remain  in  the  stain  from  24  to  48 
hours,  are  then  well  washed  in  flowing  water,  dehydrated 
in  graded  alcohol,  embedded  in  paraffin  or  celloidin.  Em- 
bryos'are  well  stained  after  this  method. 

Professor  Gibbes1  hcBmatoxylin  solution.* — One  pound 
of  logwood  chips  is  mixed  in  a  granite  kettle  with  50 
ounces  uf  distilled  water.  Bring  slowly  to  boiling  point 
and  then  allow  to  boil  for  10  minutes ;  add  about  an  ounce 
of  potash  alum  and  boil  10  minutes  longer,  constantly  stir- 
ring. Allow  to  cool  and  filter  at  the  end  of  24  hours  and 
add  ten  ounces  of  alcohol.  Before  using  dilute  with  dis- 
tilled water ;  about  10  drops  of  the  stain  are  filtered  into  a 
watch-crystal  full  of  water.  Sections  stain  for  15  minutes, 
are  then  washed  in  filtered  ''  tap-water;  "  dehydrated  in 
alcohol ;  cleared  and  mounted  in  balsam. 

Haidenhain'*  s  hcernatoxylin  solution. — Tissues  need  to 
be  hardened  in  alcohol,  and  stained  in  mass.  Gland  tis- 
sues are  well  stained  after  this  method.  Small  pieces  of 
the  tissue  are  placed  in  a  one  per  cent,  watery  solution  of 
haematoxylin  crystals,  in  this  they  remain  from  eight  to 
twelve  hours.  They  are  then  transferred  to  a  one  per 

*  Practical  Pathology.    Pago  42. 


—  109  — 

cent,  watery  solution  of  bichromate  of  potash  from  12  to 
18  hours  ;  in  (his  solution  the  tissues  become  jet  black. 
Embed  in  paraffin. 

Weiyerfs  hc&matoxylin  solution.  —  Used  for  staining 
the  central  nervous  system.  Tissues  need  to  be  hardened 
in  Midler's  fluid,  transferred  wilhout  washing  into  alcohol 
and  embedded  in  celloidin.  The  celloidin  block  is  placed 
in  the  following  solution  of  acetate  of  potash  from  24  to 
48  hours. 

Acetate  potash  (saturated  watery  solution)) 


Distilled  water  f 

The  block  is  then  washed  in  70  percent,  alcohol  for  24 
hours  and  may  then  be  sectioned.  (If  desired  the  sections 
may  first  be  cut  and  then  placed  in  the  above  solution  of 
acetate  of  potash  for  24  hours  and  washed  for  several  hours 
in  70  per  cent,  alcohol).  Sections  are  now  stained  in 
Weigert's  hrematoxylin  solution  : 

Ilaematoxylin  crystals        .....  1  grin. 

Absolute  alcohol         ......  10  c.  cm. 

Lithium  carbonate      ......  1.2  grm«. 

Distilled  water             ......  100  c.  cm. 

The  baeraatoxylin  crystals  are  dissolved  in  the  abso- 
lute alcohol,  the  lithium  carbonate  in  the  water—  mix  the 
two  solutions.  The  sections  remain  in  the  stain  12  to  24 
hours,  are  then  washed  in  Weigert's  differentiating 
fluid  : 

Borax      ...        .        .         .        .        .        .2  grms. 

Potassium  ferricyanide          .        .         .         .         .  2.  5  grms. 

Distilled  water       ......          100  c.  cm. 

In  this  solution  the  sections  remain  until  the  gray 
matter  is  clearly  u  mapped  out."  Wash  in  water—  dehy- 
drate ;  clear  in  oil  of  bergamot  ;  mount  in  bals;im.  This 
method  has  been  variously  modified  by  a  number  of  inves- 
tigators, the  most  important  of  which  is  the  modification 
recommended  by  Pal. 


- 110  — 

Pal  washes  the  sections,  after  they  have  been  stained 
in  Weigert's  hrematoxylin  solution,  first  in  a  0.25%  watery 
solution  of  permanganate  of  potassium,  in  which  the  sec- 
tions remain  for  20  to  30  minutes,  or  until  the  gray  matter 
can  be  distinguished  from  the  white,  and  completes  the 
differentiation  in  the  following  solution  : 

Oxalic  acid, 1  grm. 

Sulphite  potassium,  (K   SOs)            .  '     .        .  1  grin. 

Distilled  water, 200  c.  cm. 

In  this  the  sections  remain  only  a  few  minutes  or 
until  the  gray  matter  has  taken  a  yellowish  color.  Wash 
in  water — dehydrate;  clear  in  oil  of  bergamot ;  mount  in 

balsam. 

CARMIN. 

Carmin  has  for  many  years  held  a  prominent  place 
among  the  stains  used  for  coloring  tissue.     It  is  especially 
useful  for  staining  them  in  bulk. 
Qrenacher^s  borax-carmin  solution. 

Carmin, 3  grms. 

Borax, .        .         4  grms. 

Distilled  water, 100  c.  cm. 

Alcohol,  (70%) 100  c.  cm. 

The  carmin  and  borax  are  dissolved  in  warm  distilled 
water,  allow  to  cool  and  add  the  alcohol;  at  the  end  of  48 
hours,  the  solution  is  filtered,  the  filtrate  must  stand  for 
several  weeks  before  using.  Tissues  hardened  in  alcohol 
or  bichloride  of  mercury  are  stained  well  after  this  method. 
The  pieces  remain  in  the  stains  from  24  to  48  hours,  are  then 
washed  in  an  "  acid  alcohol  wash  "  (6  to  8  drops  of  HC1  to 
100  c.  cm.  of  10%  alcohol)  from  6  to  24  hours,  and  then  in 
70%  alcohol  for  two  hours,  dehydrated  in  alcohol  and  em- 
bedded in  celloidin  or  paraffin. 
Grenadiers  alum-carmin  solution. 

Carmin, 1  grm. 

Potash  alum,  3  grms. 

Distilled  water,        .  .  100  c.  cm. 


—  Ill  — 

Add  the  alum  and  carmin  to  the  water  ;  place  over  a 
rlame  and  bring  the  water  to  the  boiling  point;  allow  to  boil 
15  minutes.  As  soon  as  the  solution  is  cold,  filter  and 
it  will  be  ready  for  use.  Tissues  hardened  in  the 
'•chrome  salts"  are  well  stained  in  this  solution;  stain  in 
mass.  The  pieces  remain  in  the  stain  from  24  to  48  hours 
(even  longer  if  they  be  large),  are  then  washed  for  several 
hours  in  flowing  water— dehydrated  ;  embedded  in  par- 
affin or  celloidin. 

Orttis  lithium  carmin  solution. 

Carmin, 2.5  grms. 

Lithium  carbonate,         .....  1.2  grms. 

Distilled  water, 100  c.  m. 

The  carmin  and  lithium  carbonate  are  dissolved  in 
warm  water;  allow  to  cool  and  filter.  Sections  remain 
in  the  stain  10  to  15  minutes,  are  then  washed  in  "  acid 
alcohol ''  or  in  TO  per  cent,  alcohol  to  which  a  few  crystals 
of  picric  acid  have  been  added;  remove  acid  by  transfer- 
ring sections  to  70  per  cent  alcohol.  Dehydrate — clear  in 
oil  ot'  bergamot;  mount  in  balsam. 

Orttis picro-lithion-carmin  solution. 

To  three  parts  of  the  above  lithium-carmin  solution 
add  one  part  of  a  saturated  watery  solution  of  picric  acid. 
Sections  stain  in  10  to  20  minutes.  Wash  in  70  per  cent, 
alcohol  to  which  a  few  crystals  of  picric  acid  have  been 
added. 

THE  ANILINE  DYES. 

It  would  lead  me  far  beyond  the  scope  of  these  notes, 
to  mention  even  very  briefly,  the  various  aniline  dyes 
that  have  found  their  way  into  the  histological  technic. 
Only  a  very  limited  number  and  such  as  have  proved  them- 
selves most  useful  are  here  mentioned.  The  tissues  need 
to  be  stained  in  sections.  They  nearly  all  stain  diffusely, 
and  need  therefore  to  be  washed  in  a  differentiating  fluid, 
this  is  usually  strong  or  acidulated  alcohol. 


—  112- 

A  solution  of  methylen  blue,  methylen  grun,  Ijismark 
brown,  dahlia,  acid  fuchsin,  nuigrosin,  gentian  violet, 
magdala  roth,  malachit  gniii  or  eosin  may  be  made  after 
the  following  formula : 

Methylen  blue  (or  any  other  of  the  above  stains)  1  grm. 
Absolute  alcohol,  .  .  .  ,  .  .  15  c.  cm. 
Distilled  water,  .  .  .  .  .  .  85  c.  cm. 

The  sections  remain  in  the  stain  five  to  thirty  minutes, 
are  then  rinsed  in  distilled  water  and  transferred  to  95  per 
cent,  alcohol,  in  which  they  remain  until  no  more  stain  is 
given  off  from  the  section.  Dehydrate  in  absolute  alcohol 
and  mount  in  balsam. 

Flemings  Safranin  solution. 

Safranin,  .......  1  grm. 

Alcohol  absolute,     ....  .  100  c.  cm. 

Distilled  water, 200  c.  cm. 

Tissues  should  be  hardened  in  Fleming's  or  Her- 
mann's solution.  Sections  remain  in  stain  24  to  48  hours. 
They  are  then  first  washed  in  hydrochloric  or  picric  acid 
alcohol  (95  per  cent,  alcohol,  to  200  c.  cm.  of  which  one 
drop  of  hydrochloric  acid  or  several  crystals  of  picric  acid 
have  been  added),  then  in  absolute  alcohol  and  mounted 
in  balsam.  Safranin  stains  especially  well  the  chromatic 
substance  of  the  nucleus,  and  is  largely  used  for  staining 
cells  in  process  of  division. 


DOUBLE  STAINING. 


When  certain  colors  are  combined  in  a  solution,  or 
n?ed  one  after  the  other  in  staining,  it  has  been  found  that 
some  elements  of  the  tissues  to  be  stained,  are  colored  by 
one  of  the  dyes  used,  while  others  show  greater  affinity  for 
the  other.  This  fact  is  made  use  of  in  combining  dyes  for 
double  and  treble  staining. 

H&matoxylin  and  eosin  or  acid  fiichsin.  Sections 
are  stained  first  in  a  haematoxylin  solution,  that  of  Boehmer, 
Khrlich,  Delatield  or  Gibbes  may  be  used.  The  hasma- 
toxylin  stained  sections  must  go  from  distilled  water  into 
the  eosin  or  acid  fnchsin  solution.  The  eosin  or  acid  fuch- 
sin  are  used  in  a  one  per  cent,  solution.  In  this  the  sec- 
tions remain  one  to  three  minutes ;  are  then  washed  first 
in  distilled  water,  then  in  95  percent,  alcohol  until  no  more 
of  the  stain  (eosin  on  acid  fuchsin)  is  given  off.  Dehy- 
drate— clear  and  mount  in  balsam.  All  nuclei  are  stained 
blue,  the  protoplasm  of  cells  and  the  connective  tissue 
red. 

Orang  G.  hwmatoxylin  (Rawitz).  The  tissues  must 
be  hardened  in  alcohol,  bichloride  of  mercury  or  picric 
acid.  Sections  are  placed  in  a  saturated  watery  solution 
of  Orang  G.  24  hours,  washed  in  distilled  water  two  to  five 
minutes,  then  stained  in  Boahmer's  hsematoxylin. 

Safranin  and  licht  grun,  (Benda,)  an  aniline  water 
solution  of  safranin  is  made  after  the  following  formula  : 

Safranin,        .        .        .     .  .        ....      1     grm. 

Aniline  water, 90  c.  cm. 

Absolute  alcohol, 10  c.  cm. 


—  114  — 

Aniline  water  is  made  by  saturating  distilled  water 
with  aniline  oil  and  filtering,  add  the  alcohol  and  the 
safranin.  Sections  remain  in  the  slain  24  hours,  are  then 
washed  in  absolute  alcohol  for  one  minute,  and  placed 
from  30  to  45  seconds  in  a  one  per  cent,  alcoholic  solution 
of  licht  griln  ;  wash  again  in  absolute  alcohol.  Clear  in  oil 
of  bergamot  and  mount  in  balsam.  The  licht  griin  removes 
the  safranin  more  quickly  from  the  protoplasm  of  the  cells 
than  from  the  chromatin  of  the  nucleus.  When  the  stain 
is  properly  managed  the  chromatin  is  stained  red,  the 
protoplasm  green. 

Eosin  and  Nigrosin,  This  method  is  useful  for  stain- 
ing sections  of  the  central  nervous  system.  Sections  are 
stained  for  one  hour  in  a  one  per  cent,  solution  of  nigrosin, 
are  then  washed  in  distilled  water  for  several  minutes  ; 
they  are  then  placed  in  a  one  per  cent,  solution  of  eosin 
in  which  they  remain  from  three  to  five  minutes. 

Wash  first  in  seventy  percent,  alcohol  then  in  95  per 
cent,  until  no  stain  is  given  off  from  the  section. 


TREBLE  STAINING. 


The  Jormula  here  given  was  first  recommended  by 
Ehrlich,  and  used  for  staining  blood  preparations.  Biondi 
and  Heidenhain  have  so  modified  it,  that  it  can  be  used 
for  staining  sections. 

Acid  fuchsin  (saturated  watery  solution),  .  20  parts. 
Methylengriin  (saturated  watery  solution),  .  50  parts. 
Orang  G.  (saturated  watery  solution),  .  .  100  prast. 

The  acid  fuchsin  and  Orang  G.  are  placed  in  a  bottle 
and  mixed,  the  rnethylengriin  is  aided  slowly,  while  con- 
stantly shaking.  The  stain  should  not  be  filtered,  the  re- 
quired amount  is  pipetted  from  the  bottle.  Before  using 
dilute  forty  to  sixty  times  with  distilled  water.  Tissues 
need  to  be  hardened  in  alcohol  or  bichloride  of  mercury. 
Sections  remain  in  the  stain  from  twelve  to  twenty-four 
hours,  are  then  washed  in  alcohol,  cleared  in  oil  of  cloves 
and  mounted  in  balsam. 


METHODS  FOR  PREPARING  AND  STAINING 
FOOD  PREPARATION. 


The  steps  for  obtaining  blood  preparations  are  the  fol- 
lowing :  Have  before  you  a  piece  of  filter  paper  on  which 
are  placed  a  number  of  carefully  cleaned  cover-glasses, 
these  must  be  very  thin,  "extra  number  one."  Ehrlioh 
washes  the  cover  glasses  first  in  strong  sulphuric  acid, 
rinses  in  water  and  places  them  for  a  few  moments  in 
glacial  acetic  acid;  they  are  then  washed  in  flowing  water 
until  all  the  acid  is  removed,  transferred  to  95  per  cent, 
alcohol,  from  which  they  are  taken  and  wiped.  Unless  the 
cover-glasses  are  thin  and  clean,  no  good  preparation  can 
be  made.  The  finger  is  pricked  with  a  steel  pen,  one  of 
the  prongs  of  which  have  been  broken  off.  From  the  flow- 
ing blood  a  very  small  drop  is  caught  on  the  cover-glass;, 
near  its  edge,  and  the'glass  quickly  placed,  blood  side  down- 
ward, on  another  cover-glass,  care  being  taken  to  cover  this 
second  cover-glass  only  about  half.  The  blood  will  be  seen 
to  spread  out  between  the  two  covers.  Quickly  draw  one 
cover-glass  from  the  other;  a  thin  layer  of  blood  will  in 
this  way  be  spread  on  both  slips.  Ten  to  twenty  prepara- 
tions are  to  be  made  in  this  way  and  placed,  blood  side  up, 
on  the  filter  paper  before  you  and  allowed  to  dry.  The 
blood  preparations  may  be  fixed  by  placing  them  in  a 
solution,  composed  of  equal  parts  of  absolute  alcohol  and 
ether,  in  which  they  remain  for  an  hour  as  suggested  by 
Nikiforoff  or  as  Ehrlich  recommends,  by  exposing  them  to 
a  temperature  of  100°  to  130°  0.  for  one-half  to  two  hours. 

Ehrlich  has  a  very  simple  apparatus,  shown  in  Fig.  2, 
for  fixing  blood  preparations.  It  consists  of  a  copper  plate 


-=-117   — 

II)  about  15  inches  long,  four  wide  and  one  eighth  inch 
thick.  The  copper  plate  is  heated  at  one  end  with  an 
alcohol  or  gas  flame.  11'  then,  at  the  end  of  15  minutes  a 

100.  C.  ('• 


A  . 

1 


Fig.  2.    plate  for  heating  blood  preparations.     (A)  copper  plate;   (C) 
blood  preparations.     F.    Tripod. 

glass  rod  which  has  been  dipped  into  water  be  passed  over 
the  plate,  beginning  at  the  end  away  from  the  flame,  a 
place  is  reached  where  the  water  begins  to  boil  ;  this  region 
of  the  copper  plate  is  looked  upon  as  having  a  temperature 
of  100°  0.,  it  is  represented  by  a  dotted  line  in  the  dia- 
gram. The  blood  preparations  (C)  are  placed  on  the 
plate  ( blood  side  up)  between  the  flame  and  this  imaginary 
line,  nearer  the  latter,  and  heated  for  a  time  differing  with 
the  stain  used. 

After  the  preparations  are  fixed  they  are  stained  in 
oner  of  the  followin    solutions  : 


neutrophile  mixture — 

Orang  G.  (saturated  aqueous  solution),     .     120-135  c.  cni. 
Acid  fuchsin  (saturated  aqueous  solution),      80-165  c.  cm. 
Methylengiun  (saturated  aqueous  solution),          125  c.  cm. 
Distilled  water,    .         .....  300  c.  cm. 

Alcohol  absolute,          .  200  c.  cm. 

Glycerin,       .......  100  c.  cm. 

Mix  orang  G.  acid  fuchsin,  water  and  absolute  alcohol 
in  a  bottle,  add  slowly  and  while  shaking  the  methylen- 
giiui.  The  glycerin  is  then  added  For  staining  in 


Ehrlich's  neutrophile mixture,  blood  preparations  need  to 
be  fixed  at  a  temperature  from  100°  to  110°  C.  for  15  to  30 
minutes.  Float  the  preparation  on  a  small  quantity  of  the 
stain  for  about  15  minutes,  wash  off  the  stain  in  flowing 
water,  dry  the  preparation  between  several  filter  papers, 
and  mount  in  balsam.  The  red  corpuscles  should  have  a 
reddish  brown  color  (brick  color),  all  nuclei  green,  the 
eosinophile  granules  red,  the  neutrophile  granules  violet. 

Chenzinski's  solution — 

Methylrn  blue  (saturated  watery  solution),  .  40  parts. 
Eosin  (1  per  cent.  sol.  in  70  per  cent,  alcohol),  .  20  parts. 

Distilled  water, 30  parts. 

Glycerin,        .        .        .        .        .  .        .10  parts. 

The  eosin,  distilled  water  and  glycerin  are  mixed  in 
a  bottle,  the  methylen  blue  is  added  slowly  while  shak- 
ing. Preparations  need  to  be  fixed  from  one  to  one  and 
one-half  hours  at  a  temperature  of  120°  C.  They  remain 
in-the  stain  for  24  hours  in  the  warm  oven  at  a  tempera- 
ture of  40°  C.  Wash  quickly  in  flowing  water,  dry 
between  filter  paper  and  mount  in  balsam.  The  red  cor- 
puscles and  the  eosinophile  granules  are  stained  red,  all 
nuclei  blue. 

EhrlicKs  triacid  glycerin  mixture — 

Aurantia,  .        . 2  grms. 

Eosin,       .  •:  ,     •        •         •         •         •         •  3  grms. 

Nigrosin, 5  grms. 

Glycerin, 40  c.  cm. 

The  glycerin  is  divided  into  three  parts,  to  each  is 
added  one  of  the  above  stains,  and  each  needs  to  be  ground 
in  a  mortar  for  several  hours.  The  three  glycerin  solu- 
tions are  then  mixed  and  exposed  to  a  temperature  of  60° 
C.  for  two  weeks.  The  stain  is  then  ready  for  use  and  if 
well  made  amply  repays  all  the  trouble  taken  in  making 
it.  It  will  keep  for  a  long  time  and  should  be  of  a  syrupy 


—  119  — 

consistancy.  The  blood  preparations  need  to  be  fixed  at  a 
temperature  of  130*  to.  140°  C.  for  one  to  two  hours.  A  small 
quantity  of  the  stain  is  spread  out  in  a  flat  dish,  on  this 
the  preparation^  are  placed,  they  remain  in  the  warm  oven 
(40°  C.)  for  24  hours;  are  then  washed  in  flowing  water, 
dried  between  filter  paper  and  mounted  in  balsam.  The 
red  blood  cells  are  stained  yellow,  all  nuclei  black,  the 
eosinophile  granules  red,  other  granules  are  not  stained. 

Ehrlic/is  methylen  blue  solution  for  staining;  baso- 
phile  cells  (Mastzellen  of  Ehrlich). 

Methylen  blue  (saturated  alcoholic  solution),     .     1  part. 
Distilled  water, 2  parts. 

Preparations  are  fixed  at  a  temperature  of  110°  C.  for  30 
minutes.  Stain  for  15  minutes.  Wash  quickly  in  flowing 
water,  dry  between  filter  paper  and  mount  in  balsam.  All 
nuclei  are  stained  blue,  and  only  the  basophile  granules, 
which  also  take  a  blue  color,  are  stained. 

The  methods  here  given  are  especially  useful  for 
studying  the  blood  clinically,  and  can  not  be  too  warmly 
recommended. 


* 


